Upload
phunglien
View
215
Download
0
Embed Size (px)
Citation preview
1
LLM-domain containing B-GATA factors control different aspects of 1
cytokinin-regulated development in Arabidopsis thaliana 2
Quirin L Ranftl1 Emmanouil Bastakis1 Carina Klermund1 and Claus 3
Schwechheimer12 4
5
Plant Systems Biology 6
Wissenschaftszentrum Weihenstephan 7
Technische Universitaumlt Muumlnchen 8
Emil-Ramann-Strasse 8 9
85354 Freising 10
Germany 11
12
One sentence summary Arabidopsis LLM-domain B-GATA transcription 13
factors control greening hypocotyl elongation phyllotaxy floral organ 14
initiation branching flowering time and senescence 15
16
Footnotes This work was supported by grants from the Deutsche 17
Forschungsgemeinschaft to CS (SCHW7519-1 and SCHW75110-1 in the 18
SPP1530) CS would like to thank the Nirit and Michael Shaoul Fund for 19
Visiting Scholars and Fellows for financial support during manuscript 20
preparation 21
22
Corresponding author Claus Schwechheimer 23
clausschwechheimerwzwtumde 24
25
Author contributions QLR and CS designed all experiments and wrote 26
the paper QLR performed all experiments and data analysis apart from the 27
microarray and the ChIP-seq analyses QLR and CK performed and 28
analyzed the microarray experiment EB and CS designed the ChIP-seq 29
experiment EB performed and analyzed the ChIP-seq experiment 30
Plant Physiology Preview Published on February 1 2016 as DOI101104pp1501556
Copyright 2016 by the American Society of Plant Biologists
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
2
ABSTRACT 31
LLM-domain B-GATAs are a subfamily of the thirty-membered GATA 32
transcription factor family from Arabidopsis thaliana Only two of the six 33
Arabidopsis LLM-domain B-GATAs GNC (GATA NITRATE-INDUCIBLE 34
CARBON METABOLISM-INVOLVED) and its paralog GNLCGA1 (GNC-35
LIKECYTOKININ-RESPONSIVE GATA FACTOR1) have already been 36
analyzed with regard to their biological function Together GNC and GNL 37
control germination greening flowering time and senescence downstream 38
from auxin CK (cytokinin) gibberellin and light signaling Whereas 39
overexpression and complementation analyses suggest a redundant 40
biochemical function between GNC and GNL nothing is known about the 41
biological role of the four other LLM-domain B-GATAs GATA15 GATA16 42
GATA17 and GATA17L (GATA17-LIKE) based on loss-of-function mutant 43
phenotypes Here we examine insertion mutants of the six Arabidopsis B-44
GATA genes and reveal the role of these genes in the control of greening 45
hypocotyl elongation phyllotaxy floral organ initiation accessory meristem 46
formation flowering time and senescence Several of these phenotypes had 47
previously not been described for the gnc and gnl mutants or were enhanced 48
in the more complex mutants when compared to gnc gnl mutants Some of 49
the respective responses may be mediated by CK signaling which activates 50
the expression of all six GATA genes CK-induced gene expression is partially 51
compromised in LLM-domain B-GATA mutants suggesting that B-GATA 52
genes play a role in CK responses We furthermore provide evidence for a 53
transcriptional cross-regulation between these GATAs that may in at least 54
some cases be at the basis of their apparent functional redundancy 55
56
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
3
INTRODUCTION 57
Transcription factors of the GATA family contain a type IV zinc finger DNA-58
binding domain (C-X2-C-X17-20-C-X2-C) which is predicted to bind the 59
consensus DNA sequence WGATAR (where W is T or A and R is G or A) 60
(Reyes et al 2004) The genomes of higher plants encode approximately 30 61
GATA transcription factors which are classified as A- B- C- and D-GATAs 62
according to their primary amino acid sequence the conservation of their 63
GATA DNA-binding domains the conservation of protein domains outside of 64
the GATA DNA-binding domain and their exon-intron structures (Reyes et al 65
2004) We recently showed that the Arabidopsis thaliana B-GATAs can be 66
further subdivided into two structural subfamilies with either a HAN- or an 67
LLM-domain located N- or C-terminally of the GATA DNA-binding domain 68
(Behringer et al 2014 Behringer and Schwechheimer 2015) The HAN-69
domain was initially identified as a conserved domain in the floral morphology 70
regulator HAN (HANABA TARANU) (Zhao et al 2004 Nawy et al 2010 71
Zhang et al 2013) the LLM-domain was named after the invariant leucine-72
leucine-methionine (LLM) motif in the conserved C-terminus of the respective 73
B-GATAs (Richter et al 2010 Behringer et al 2014) 74
A recent comparative analysis of B-GATAs from Arabidopsis Solanum 75
lycopersicon (tomato) Hordeum vulgare (barley) and Brachipodium 76
distachyon (Brachipodium) revealed that HAN-domain and LLM-domain B-77
GATAs have different biological and biochemical activities the expression of 78
HAN-domain B-GATAs under the control of an LLM-domain B-GATA 79
promoter could not complement LLM-domain B-GATA loss-of-function 80
mutants and the overexpression of HAN- and LLM-domain B-GATAs in 81
Arabidopsis differentially affected plant growth (Behringer et al 2014 82
Behringer and Schwechheimer 2015) The existence of the Brassicaceae-83
specific GATA23 which has a degenerate LLM-domain as well as the 84
existence of monocot-specific HAN-domain B-GATAs reveal that expansions 85
of the B-GATA family have been used during plant evolution to control specific 86
aspects of plant development (De Rybel et al 2010 Whipple et al 2010 87
Behringer and Schwechheimer 2015) 88
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
4
The Arabidopsis LLM-domain B-GATA family is comprised of GATA15 89
GATA16 GATA17 and GATA17L (GATA17-LIKE) as well as GNC (GATA 90
NITRATE-INDUCIBLE CARBON METABOLISM-INVOLVED GATA21 91
hitherto GNC) and GNL (GNC-LIKECYTOKININ-RESPONSIVE GATA 92
FACTOR1 hitherto GNL) (Behringer et al 2014 Behringer and 93
Schwechheimer 2015) Among these the paralogous GNC and GNL are 94
already well characterized with regard to their biological function and their 95
upstream regulation gnc and gnc gnl mutants are defective in greening and 96
chloroplast biogenesis and they germinate and flower slightly earlier than the 97
wild type (Bi et al 2005 Richter et al 2010 Chiang et al 2012 Richter et 98
al 2013) Conversely overexpression of the two GATAs results in a strong 99
increase in chlorophyll accumulation and delayed germination and flowering 100
(Richter et al 2010 Richter et al 2013 Behringer et al 2014) In addition 101
GNC and GNL overexpression promotes hypocotyl elongation in light-grown 102
seedlings alterations in leaf shape and an increased angle between the 103
primary and lateral inflorescences (Behringer et al 2014) Importantly 104
deletion or mutation of the LLM-domain of GNC or GNL compromises some 105
but not all phenotypes when these deletion or mutant variants are 106
overexpressed in Arabidopsis and the effects of the overexpression 107
transgenes are compared to the effects of the overexpression of the wild type 108
proteins (Behringer et al 2014) The fact that the overexpression of every 109
LLM-domain B-GATA factor tested to date results in highly comparable 110
phenotypes suggested that LLM-domain B-GATAs have conserved 111
biochemical functions (Behringer et al 2014) 112
A range of studies have already addressed the upstream signaling pathways 113
controlling GNC and GNL expression GNC was initially identified as a nitrate-114
inducible gene with an apparent role in the control of greening (Bi et al 2005) 115
GNL was originally identified based on its strong CK- (cytokinin-)regulated 116
gene expression and therefore designated CYTOKININ-RESPONSIVE GATA 117
FACTOR1 (CGA1) (Naito et al 2007) The expression of GNC and GNL is 118
also controlled by the DELLA regulators of the GA (gibberellin) signaling 119
pathway as well as by the light-labile PIFs (PHYTOCHROME INTERACTING 120
FACTORS) of the phytochrome signaling pathway which are themselves 121
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
5
negatively regulated by DELLAs (Richter et al 2010 Leivar and Monte 122
2014) Furthermore the auxin signaling regulators ARF2 (AUXIN RESPONSE 123
FACTOR2) and ARF7 the flowering regulator SOC1 (SUPPRESSOR-OF-124
OVEREXPRESSION-OF-CONSTANS1) as well as the floral development 125
regulators AP3 (APETALA3) and (PI) PISTILLATA have been implicated in 126
GNC and GNL transcription control (Mara and Irish 2008 Richter et al 2010 127
Richter et al 2013 Richter et al 2013) Thus multiple signaling pathways 128
converge on GNC and GNL expression and the two genes control a broad 129
range of developmental responses in plants 130
To date nothing is known about the regulation and biological function of the 131
four Arabidopsis LLM-domain B-GATAs GATA15 GATA16 GATA17 and 132
GATA17L Here we examine the entire LLM-domain B-GATA gene family 133
through the analysis of single gene insertion mutants and a range of mutant 134
combinations These analyses reveal mainly overlapping roles for these B-135
GATAs in the control of previously known B-GATA-regulated processes such 136
as greening hypocotyl elongation flowering time and senescence but they 137
also reveal previously unknown functions of LLM-domain B-GATAs in 138
phyllotactic patterning floral organ initiation and accessory meristem 139
formation Several of these responses may be mediated by CK signaling and 140
CK treatments induce the expression of all six GATA genes CK-induced gene 141
expression is partially compromised in LLM-domain B-GATA mutants 142
suggesting that B-GATA genes play a role in CK responses Furthermore we 143
provide evidence for a cross-regulation of the different LLM-domain B-GATA 144
genes at the transcriptional level 145
146
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
6
RESULTS 147
Evolutionary conservation of the LLM-domain B-GATAs within the 148
Brassicaceae The Arabidopsis thaliana genome encodes six LLM-domain 149
GATA transcription factors namely GNC (AtGATA21) GNL (AtGATA22) as 150
well as GATA15 (AtGATA15) GATA16 (AtGATA16) GATA17 (AtGATA17) 151
and GATA17L (AtGATA17-LIKE) (Behringer et al 2014 Behringer and 152
Schwechheimer 2015) Phylogenetic analyses showed that these proteins 153
can be grouped into three pairs of highly homologous proteins comprised of 154
the with regard to the length of their N-termini long B-GATAs GNC and GNL 155
and the short B-GATAs GATA15 and GATA16 as well as GATA17 and 156
GATA17L respectively (Fig 1A and B Fig S1) (Behringer et al 2014 157
Behringer and Schwechheimer 2015) It had previously become apparent 158
that the respective subfamilies of long and short B-GATAs were conserved in 159
different plant species but that gene duplications in individual subfamilies may 160
have led to a differential expansion of these subfamilies in different species 161
(Behringer et al 2014) To gain an insight into the conservation of these 162
GATAs within the family of Brassicaceae we searched for LLM-domain B-163
GATAs in the fully sequenced genomes of Arabidopsis lyrata Capsella 164
rubella Eutrema salsugineum and Brassica rapa We retrieved orthologs for 165
each of the six Arabidopsis B-GATAs within each Brassicaceae species (Fig 166
S1 and Fig S2) Whereas the Arabidopsis lyrata Capsella rubella and 167
Eutrema salsugineum genomes contained exactly one apparent ortholog for 168
each of the six Arabidopsis thaliana genes the Brassica rapa genome known 169
to have undergone hexaploidization followed by genome fractionation 170
retained at least two orthologs each for GNC GNL GATA17 and GATA17L 171
and one ortholog of GATA15 and GATA16 (Fig S1 and Fig S2) (Wang et al 172
2011 Tang et al 2012) In summary we concluded that the six LLM-domain 173
B-GATAs are conserved in the different Brassicaceae examined here and that 174
four members of the gene family were present in multiple copies in Brassica 175
rapa 176
177
Characterization of insertion mutants of the six LLM-domain B-GATA 178
genes All previously published genetic analyses of the LLM-domain B-179
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
7
GATAs genes from Arabidopsis thaliana focused on GNC and GNL 180
(Behringer and Schwechheimer 2015) As yet no mutants have been 181
described for the remaining four LLM-domain B-GATAs and their biological 182
function remains elusive We therefore identified homozygous lines of T-DNA 183
insertion mutants for each of the six genes (Fig 1C Fig S3) and 184
subsequently analyzed these lines with regard to the effects of the respective 185
mutations on gene expression using quantitative real time PCR (qRT-PCR) 186
and semi-quantitative PCR (sqRT-PCR) (Fig S3B) The analysis of the gnc 187
allele (Salk_001778) which had already been used in several publications 188
suggested that neither the full-length transcript nor regions downstream from 189
the insertion site were transcribed (Fig 1C and Fig S3B) (Richter et al 2010) 190
(Bi et al 2005) Similarly analyses of the gnl insertion mutant (Salk_003995) 191
using primers spanning the entire transcript or flanking the T-DNA harbored in 192
the intron suggested that the GNL full-length transcript was absent (Fig 1C 193
and Fig S3B) (Bi et al 2005 Richter et al 2010) Although one of the 194
selected GATA15 insertion alleles (gata15-1 SAIL_618_B11) was annotated 195
as an in-gene insertion (wwwsignalsalkedu) we found no evidence for a 196
change of GATA15 gene expression in the homozygous insertion line (Fig 1C 197
and Fig S3B) Sequencing of the insertion site then revealed that the 198
insertion was downstream from the genersquos 3-UTR (untranslated region) 199
indicating that the GATA15 gene was intact in SAIL_618_B11 (Fig 1C and 200
Fig S3B) The analysis of a second allele (gata15-2 WiscDsLox471A10) with 201
an exon-insertion revealed a strong down-regulation of GATA15 expression in 202
both RT-PCR analyses (Fig 1C and Fig S3B) In a GATA16 allele with a T-203
DNA insertion in the genersquos 3-UTR (gata16-1 Salk_021471) transcript 204
abundance was partially reduced (Fig 1C and Fig S3B) The abundance of 205
GATA17 was also partially reduced in Salk_101994 (gata17-1) which 206
harbored a T-DNA insertion in the genes 5-UTR and strongly reduced in 207
SALK_049041 (gata17-2) which carried an insertion in the LLM-domain-208
encoding gene region (Fig 1C and Fig S3B) Finally we detected no 209
transcript in our analyses of a GATA17L exon insertion allele (gata17l-1 210
Salk_026798) when using primer pairs spanning the insertion or located 211
downstream from the insertion site (Fig 1C and Fig S3B) When analyzing 212
this allele with a primer combination upstream of the insertion we were able 213
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
8
to detect residual transcript in gata17l-1 but judged that this may not give rise 214
to a fully functional protein since the insertion was upstream of the LLM-215
domain (Fig 1C and Fig S3B) We concluded that the expression of the 216
respective GATA genes was absent or at least strongly reduced in alleles for 217
GNC (gnc) GNL (gnl) GATA15 (gata15-2) GATA17 (gata17-2) and 218
GATA17L (gata17l-1) alleles The gata16-1 and gata17-1 alleles were only 219
partially impaired in the expression of the respective genes whereas the 220
gata15-1 allele was not affected in GATA15 expression The gata15-2 and 221
gata17-2 alleles became available to us only later in this study and for 222
strategic reasons could not be included in the generation of the complex 223
mutant combinations described below Any of the phenotypes described in the 224
following sections for the more complex mutant combinations were not 225
apparent in the gata15-2 and gata17-2 single mutants We therefore exclude 226
the possibility that these genes are by themselves responsible for any of the 227
phenotypes described here for the higher order mutants 228
229
LLM-domain B-GATAs redundantly control greening We did not observe 230
any obvious defects in the gata single mutants apart from the already well-231
documented defects in greening of the gnc mutants (Bi et al 2005 Richter et 232
al 2010 Chiang et al 2012) To generate higher order mutants in the 233
closely related B-GATA genes we combined the alleles gnc gnl gata15-1 234
gata16-1 gata17-1 and gata17l-1 For simplicity we will subsequently omit 235
the respective allele numbers Since the presence of the misannotated 236
gata15-1 mutation does not provide information about the loss of GATA15 we 237
refer to this allele as GATA15 238
Through genetic crosses we obtained gata17 gata17l double mutants 239
GATA15 gata16 gata17 gata17l gnc gnl GATA15 gata16 and gnc gnl gata17 240
gata17l triple and quadruple mutants as well as a gnc gnl GATA15 gata16 241
gata17 gata17l quintuple mutant In the following we will refer to the gata 242
quintuple mutant as the quintuple mutant and to the GATA15 gata16 gata17 243
gata17l triple mutant that does not include the gnc and gnl alleles as the 244
(complementary) triple mutant In all other cases we will specify the allele 245
combinations 246
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
9
To examine a possible redundant function among the B-GATAs in the control 247
of greening we analyzed greening and chlorophyll content in 14 day-old light-248
grown gata mutant plants (Fig 1D and E) Although there was no indication 249
for a defect in greening in gata17 gata17l or in the triple mutant we found that 250
the greening defect of gnc gnl was further enhanced in the gnc gnl gata17 251
gata17l quadruple mutant and even more in the quintuple mutant (Fig 1D and 252
E) At the same time greening defects were not obvious in any of the gata 253
single mutants (Fig 1E) In the case of the gata15 and gata17 alleles this 254
was not due to the fact that weak alleles had been chosen for the complex 255
mutants since also the severe alleles gata15-2 and gata17-2 showed no 256
reduction in chlorophyll levels when compared to the wild type (Fig 1E) 257
The further reduction in chlorophyll accumulation in the quintuple mutant was 258
also apparent when we analyzed chlorophyll abundance by fluorescence 259
microscopy where we found reduced chloroplast fluorescence when 260
comparing the quintuple mutant to gnc gnl (Fig 1F) Furthermore we noted 261
that the greening defect gradually disappeared in gnc gnl mutants after the 262
seedling stage but remained apparent in the quintuple mutants also in adult 263
plants (Fig S4) Taken together this suggested that all mutated GATA genes 264
participate in the control of greening and chlorophyll accumulation and that 265
the defects of mutants defective in a single gene may be suppressed by the 266
presence of the other family members 267
268
Gene expression regulation of the LLM-domain B-GATAs GNC and GNL 269
had been in the focus of several studies based on the strong regulation of 270
their gene expression by light and CK (Bi et al 2005 Naito et al 2007 271
Richter et al 2010 Richter et al 2013) We were therefore interested in 272
evaluating to what extent light and CK regulate the expression of the other 273
GATA genes In the case of light regulation we found that far-red red as well 274
as blue light strongly induced the expression of GNL and to some extent also 275
the expression of GNC (Fig 2) In blue light we noted a subtle but significant 276
induction of GATA15 and GATA16 (Fig 2C) Importantly this regulation was 277
not observed in the red and far-red light receptor mutant phyA phyB or in the 278
blue light receptor mutant cry1 cry2 (Fig 2) Furthermore light-induced gene 279
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
10
expression changes in red light were impaired in the quadruple mutant for the 280
PHYTOCHROME INTERACTING FACTOR (PIF) genes PIF1 PIF3 PIF4 281
and PIF5 (pifq) indicating that the light-dependent regulation in red light was 282
mediated by the phytochrome and PIF signaling pathways (Fig S5) (Leivar et 283
al 2008) 284
The CK experiments indicated that CK induces the expression of all six GATA 285
genes (Fig 3) With an about three-fold increase in transcript abundance this 286
induction was strongest for GNL but could be observed for each of the other 287
GATA genes after a one to four hour CK treatment (Fig 3) Thus CK 288
promotes the expression of all six LLM-domain B-GATAs 289
290
Mutants of LLM-domain B-GATAs are impaired in hypocotyl elongation 291
Due to the strong regulation particularly of GNL but also of GNC in far-red red 292
and blue light conditions we examined the contribution of the GATA genes to 293
hypocotyl elongation by analyzing mutant seedlings in the different light 294
conditions in two different light intensities (Fig 4) Here we found that 295
quintuple mutant seedlings had a shorter hypocotyl than wild type seedlings in 296
white light as well as in red far-red and blue light (Fig 4) Notably these 297
phenotypes were not apparent or at least not significant in the gnc gnl 298
double mutant or the triple mutant suggesting that the respective other GATA 299
genes may contribute to the hypocotyl elongation phenotype in these 300
backgrounds (Fig 4) At the same time hypocotyl length of dark-grown 301
mutant seedlings was indistinguishable in all genotypes from that observed in 302
the wild type (Fig 4E and G) The short hypocotyl phenotype of the GATA 303
gene mutants was also interesting because we previously observed that the 304
LLM-domain B-GATA overexpressors had a longer hypocotyl than the wild 305
type when grown in white light (Behringer et al 2014) When analyzed in the 306
different light conditions we found the long hypocotyl phenotype of the GNL 307
overexpression line (GNLox) was also present in far-red and blue light but 308
interestingly not in red light where hypocotyls were even shorter than in the 309
wild type or the mutants when grown in weak red light conditions (Fig 4) 310
311
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
11
GATA gene mutants have differential gene expression defects The 312
strong induction of GNL gene expression by CK was already repeatedly 313
described and is the reason for the genersquos original designation CGA1 314
(CYTOKININ-INDUCED GATA1) (Naito et al 2007 Kollmer et al 2011) In 315
order to examine the role of the GATA genes in CK-responsive gene 316
expression we performed microarray experiments with 14 day-old wild type 317
plants gnc gnl and triple mutants as well as quintuple mutants treated for 60 318
min with the CK 6-BA (6-benzylaminopurine) or a corresponding mock solvent 319
control (Fig 5 and Fig 6 Supplemental Tables S1 and S2) We then 320
analyzed the resulting data with regard to differences in basal gene 321
expression (Fig 5) and with regard to gene expression differences after the 322
CK treatment in comparison to the mock control (Fig 6) For technical 323
reasons the experiment was conducted in two rounds the first experiment 324
included the Col-0 (1) wild type control the gnc gnl double mutant and the 325
quintuple mutant the second experiment included an independent Col-0 (2) 326
wild type control and the triple mutant When we examined the expression of 327
the CK-induced ARRs (A-TYPE RESPONSE REGULATORS) of the CK 328
signaling pathway we could confirm that the respective CK induction 329
experiments were comparably efficient between the two experiments and in 330
all genotypes tested (Fig S6A) We also examined the microarray dataset 331
with regard to the expression of a previously defined so-called ldquogolden listrdquo of 332
CK-regulated genes (Bhargava et al 2013) Over 70 of the 331 entities 333
derived from this list were significantly CK-regulated in our experiments 334
(Supplemental Table S3) When applying a two-fold expression change as 335
additional filter criterion 71 of these 331 entities were identified as up- (Col-0 336
1 and Col-0 2) and 39 (Col-0 1) and 25 entities (Col-0 2) as down-regulated 337
after CK treatment (Fig S6B and Supplemental Table S3) We thus 338
concluded that the CK treatments in both experiments were similarly efficient 339
and that the data sets could be used for comparative analyses 340
The comparison of the basal gene expression profiles of the wild type 341
samples and the mutants indicated that the (untreated) quintuple mutant 342
seedlings had by far the largest number of differentially regulated genes with 343
4722 down-regulated and 976 up-regulated entities when compared to the 344
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
12
Col-0 (1) wild type (Fig 5 and Supplemental Table S1) Furthermore the gnc 345
gnl double mutant contained 2458 down- and 294 up-regulated entities 346
respectively (Fig 5 and Supplemental Table S1) In comparison the number 347
of differentially expressed genes was comparatively minor in the triple mutant 348
which had only 323 down- and 278 up-regulated entities in this particular 349
experiment and selected physiological growth conditions (Fig 5) 350
The comparatively strong gene expression differences in the gnc gnl double 351
and the quintuple mutants and the fact that both mutants shared the gnc gnl 352
loss-of-function at the genetic level was reflected by the large overlap 353
between the differentially expressed gene sets of the two genotypes 354
Specifically 1922 of the 2458 down-regulated entities from gnc gnl were also 355
down-regulated in the quintuple mutant (Fig 5B) and 98 of the 294 entities 356
up-regulated in gnc gnl were also up-regulated in the quintuple mutant (Fig 357
5B) Conversely the overlap in gene expression defects between the triple 358
mutant and the quintuple mutant was higher than that between the triple 359
mutant and gnc gnl (Fig 5B) We therefore concluded that the gene 360
expression differences between the different GATA gene mutants was 361
strongest in the quintuple mutant and that the strong increase in the number 362
of differentially regulated genes exceeded by far the differences seen in the 363
gnc gnl double mutant and the triple mutant This supported our conclusion 364
from the phenotypic analyses that the GATA genes act in a functionally 365
redundant manner 366
Since we had observed increased gene expression of all six GATA genes 367
after CK-treatment we expected that CK-induced gene expression may be 368
compromised in the gata mutants (Fig 6) Indeed of the 996 entities that 369
were down-regulated after CK-treatment in the wild type (Col-0 1) only 173 370
(18 ) and 89 (9 ) remained CK-regulated in the quintuple and gnc gnl 371
mutant respectively (Fig 6A and Supplemental Table S2) At the same time 372
of the 188 entities that were down-regulated after CK treatment in the wild 373
type sample of the second experiment (Col-0 2) 83 (44 ) remained CK-374
regulated in the gata triple mutant (Fig 6B) The differential effect on gene 375
expression between the different mutants was less pronounced when 376
examining the CK-induced genes Here 49 (quintuple mutant) 59 (gnc 377
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
13
gnl) and 63 (triple mutant) of the CK-induced entities from the respective 378
wild types remained CK-regulated in the mutants (Fig 6A and B) In summary 379
we concluded that CK-regulated gene expression is impaired in the GATA 380
gene mutants 381
382
GATA gene mutants are defective in CK-regulated developmental 383
processes Based on the strong defects of gata mutants in CK-regulated 384
gene expression we searched for phenotypes that could be explained by 385
defects in CK response CK signaling was described as a regulator of 386
phyllotactic patterning in the shoot apical meristem of Arabidopsis (Besnard et 387
al 2014) In line with a role of the LLM-domain B-GATA genes in this process 388
we observed frequent aberrations from normal phyllotactic patterning in flower 389
positioning in triple and quintuple mutant inflorescences (Fig 7) Quantitative 390
analyses revealed aberrations from the canonical angle of ~1375deg in about 391
~30 of the petioles in triple and quintuple inflorescences (Fig 7D) At the 392
same time this patterning defect was not apparent in the gnc gnl double 393
mutant Since we also did not observe a quantitative increase in the severity 394
of this phenotype between the triple and the quintuple mutant we concluded 395
that GATA16 GATA17 and GATA17L may be responsible for this phenotype 396
(Fig 7D) 397
Leaf senescence is another CK-regulated process (Hwang et al 2012) In the 398
wild type CK levels are reduced during senescence and positive or negative 399
interference with the decrease in CK levels can promote and delay 400
senescence respectively (Hwang et al 2012) Senescence can be induced 401
by shifting light-grown plants to the dark and we had previously already shown 402
that leaf senescence is delayed in overexpression lines of LLM-domain B-403
GATAs (Behringer et al 2014) To examine possible senescence defects in 404
the gata mutants we floated leaves of wild type and mutant plants in the dark 405
on a mock solution or on a solution containing 005 microM or 01 microM 6-BA (Fig 406
8A ndash F) (Weaver and Amasino 2001) In line with the expectation that the 407
presence of CK in the medium would suppress the senescence process we 408
observed decreased senescence in CK-treated wild type leaves which we 409
quantitatively assessed by measuring chlorophyll levels in the mock- and CK-410
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
14
treated leaves (Fig 8F) Here we found that the effects of the senescence 411
treatment were less efficiently suppressed by CK treatments in the gnc gnl 412
double and in the quintuple mutant than in the wild type or the triple mutant 413
(Fig 8A - F) Similarly we observed a stronger effect on the decreases in 414
chlorophyll and total protein levels in the gnc gnl as well as the quintuple 415
mutant when we transferred light-grown plants for four days to the dark (Fig 416
8G ndash I) As previously reported GNL overexpressing plants (GNLox) had 417
senescence phenotypes that were antagonistic to the ones observed with the 418
mutants (Fig 8) (Behringer et al 2014) In the case of the leaf floating assay 419
CK-treatment had a stronger effect on greening and chlorophyll abundance in 420
GNLox than in the wild type (Fig 8A ndash F) When we transferred plants from 421
the light to the dark GNLox seedlings remained visibly green and did not 422
display the strong decrease in total protein abundance that we observed in 423
the wild type or the GATA gene mutants (Fig 8G ndash I) We thus concluded that 424
GATA gene mutants and overexpressors have antagonistic effects with 425
regard to the regulation of senescence and that GNC and GNL contribute 426
particularly strongly to this phenotype 427
CK induces greening in light-grown seedlings (Kobayashi et al 2012) To 428
examine to what extent CK can induce chlorophyll accumulation in the gata 429
mutants we grew plants for two weeks in constant light on medium containing 430
CK (5 nM BA) or a corresponding CK-free medium As expected chlorophyll 431
content was increased in wild type plants but this increase was neither 432
significant in the gnc gnl nor the triple or the quintuple mutants (Fig S7) We 433
thus concluded that the GATA factors are required for CK-induced greening in 434
Arabidopsis seedlings 435
It was previously reported that hypocotyl elongation could be repressed by 436
incubating dark-grown seedlings in the presence of CK (Su and Howell 1995) 437
Since the GATA gene mutants had displayed a hypocotyl elongation defect in 438
light-grown seedlings (Fig 4) we also explored the link between hypocotyl 439
elongation and CK However when we tested the effects of CK on hypocotyl 440
elongation in dark-grown seedlings we did not detect any significant 441
differences in the response between the different genotypes tested (Fig S8) 442
It is thus unlikely that the GATA genes participate in this elongation response 443
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
15
Branching is another phenotype that has been attributed to defects in CK 444
biosynthesis and response (El-Showk et al 2013) When we analyzed the 445
general habitus of the quintuple mutants we noted that they had an increased 446
number of secondary branches that was combined with a reduced plant 447
height (Fig 9A - C) Occasionally we also observed the outgrowth of 448
inflorescences in the axils of lateral inflorescences in the mutants whereas we 449
had not noted this phenotype in the wild type (Fig 9D and E) A quantitative 450
analysis of these phenotypes over all gata mutant phenotypes revealed that 451
the number of first and second order lateral branches increased with 452
increasing mutant complexity (Fig 9B) Here we observed strong effects 453
already in the gata17 gata17l mutant as well as the gnc gnl double mutant but 454
strongest defects in the triple and the quintuple mutant thus all five GATA 455
genes may contribute to this phenotype (Fig 9B) At the same time plant 456
height was reduced to a significant but still minor level in these mutants 457
suggesting that decreases in plant height which may have indirect 458
consequences on the general plant habitus and branching are not the 459
primary cause for altered branching patterns in the gata mutants (Fig 9C) It 460
is further important to note that the higher order GATA gene mutants initiated 461
fewer vegetative buds but had a higher number of growing buds as reflected 462
by the increased branch numbers (Fig 9F) Thus axillary bud formation is 463
promoted and bud outgrowth is repressed by the GATAs in the wild type 464
Another phenotype that became apparent during our analysis of the gata 465
mutant plant habitus was an increase in the angle formed between the 466
primary inflorescence and the lateral inflorescences (Fig 9G and H Fig S9) 467
gata mutants had a decreased angle formed between the primary 468
inflorescence and lateral inflorescences and this phenotype was strongest in 469
the quintuple mutant and intermediate in the gnc gnl and triple mutants (Fig 470
9G and H Fig S9) Interestingly we had previously observed increased 471
branch angles in GATA overexpression lines such as GNLox (Fig S9) 472
(Behringer et al 2014) We thus concluded that GATA gene mutants and 473
overexpressors antagonistically regulate branch angles in Arabidopsis 474
475
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
2
ABSTRACT 31
LLM-domain B-GATAs are a subfamily of the thirty-membered GATA 32
transcription factor family from Arabidopsis thaliana Only two of the six 33
Arabidopsis LLM-domain B-GATAs GNC (GATA NITRATE-INDUCIBLE 34
CARBON METABOLISM-INVOLVED) and its paralog GNLCGA1 (GNC-35
LIKECYTOKININ-RESPONSIVE GATA FACTOR1) have already been 36
analyzed with regard to their biological function Together GNC and GNL 37
control germination greening flowering time and senescence downstream 38
from auxin CK (cytokinin) gibberellin and light signaling Whereas 39
overexpression and complementation analyses suggest a redundant 40
biochemical function between GNC and GNL nothing is known about the 41
biological role of the four other LLM-domain B-GATAs GATA15 GATA16 42
GATA17 and GATA17L (GATA17-LIKE) based on loss-of-function mutant 43
phenotypes Here we examine insertion mutants of the six Arabidopsis B-44
GATA genes and reveal the role of these genes in the control of greening 45
hypocotyl elongation phyllotaxy floral organ initiation accessory meristem 46
formation flowering time and senescence Several of these phenotypes had 47
previously not been described for the gnc and gnl mutants or were enhanced 48
in the more complex mutants when compared to gnc gnl mutants Some of 49
the respective responses may be mediated by CK signaling which activates 50
the expression of all six GATA genes CK-induced gene expression is partially 51
compromised in LLM-domain B-GATA mutants suggesting that B-GATA 52
genes play a role in CK responses We furthermore provide evidence for a 53
transcriptional cross-regulation between these GATAs that may in at least 54
some cases be at the basis of their apparent functional redundancy 55
56
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
3
INTRODUCTION 57
Transcription factors of the GATA family contain a type IV zinc finger DNA-58
binding domain (C-X2-C-X17-20-C-X2-C) which is predicted to bind the 59
consensus DNA sequence WGATAR (where W is T or A and R is G or A) 60
(Reyes et al 2004) The genomes of higher plants encode approximately 30 61
GATA transcription factors which are classified as A- B- C- and D-GATAs 62
according to their primary amino acid sequence the conservation of their 63
GATA DNA-binding domains the conservation of protein domains outside of 64
the GATA DNA-binding domain and their exon-intron structures (Reyes et al 65
2004) We recently showed that the Arabidopsis thaliana B-GATAs can be 66
further subdivided into two structural subfamilies with either a HAN- or an 67
LLM-domain located N- or C-terminally of the GATA DNA-binding domain 68
(Behringer et al 2014 Behringer and Schwechheimer 2015) The HAN-69
domain was initially identified as a conserved domain in the floral morphology 70
regulator HAN (HANABA TARANU) (Zhao et al 2004 Nawy et al 2010 71
Zhang et al 2013) the LLM-domain was named after the invariant leucine-72
leucine-methionine (LLM) motif in the conserved C-terminus of the respective 73
B-GATAs (Richter et al 2010 Behringer et al 2014) 74
A recent comparative analysis of B-GATAs from Arabidopsis Solanum 75
lycopersicon (tomato) Hordeum vulgare (barley) and Brachipodium 76
distachyon (Brachipodium) revealed that HAN-domain and LLM-domain B-77
GATAs have different biological and biochemical activities the expression of 78
HAN-domain B-GATAs under the control of an LLM-domain B-GATA 79
promoter could not complement LLM-domain B-GATA loss-of-function 80
mutants and the overexpression of HAN- and LLM-domain B-GATAs in 81
Arabidopsis differentially affected plant growth (Behringer et al 2014 82
Behringer and Schwechheimer 2015) The existence of the Brassicaceae-83
specific GATA23 which has a degenerate LLM-domain as well as the 84
existence of monocot-specific HAN-domain B-GATAs reveal that expansions 85
of the B-GATA family have been used during plant evolution to control specific 86
aspects of plant development (De Rybel et al 2010 Whipple et al 2010 87
Behringer and Schwechheimer 2015) 88
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
4
The Arabidopsis LLM-domain B-GATA family is comprised of GATA15 89
GATA16 GATA17 and GATA17L (GATA17-LIKE) as well as GNC (GATA 90
NITRATE-INDUCIBLE CARBON METABOLISM-INVOLVED GATA21 91
hitherto GNC) and GNL (GNC-LIKECYTOKININ-RESPONSIVE GATA 92
FACTOR1 hitherto GNL) (Behringer et al 2014 Behringer and 93
Schwechheimer 2015) Among these the paralogous GNC and GNL are 94
already well characterized with regard to their biological function and their 95
upstream regulation gnc and gnc gnl mutants are defective in greening and 96
chloroplast biogenesis and they germinate and flower slightly earlier than the 97
wild type (Bi et al 2005 Richter et al 2010 Chiang et al 2012 Richter et 98
al 2013) Conversely overexpression of the two GATAs results in a strong 99
increase in chlorophyll accumulation and delayed germination and flowering 100
(Richter et al 2010 Richter et al 2013 Behringer et al 2014) In addition 101
GNC and GNL overexpression promotes hypocotyl elongation in light-grown 102
seedlings alterations in leaf shape and an increased angle between the 103
primary and lateral inflorescences (Behringer et al 2014) Importantly 104
deletion or mutation of the LLM-domain of GNC or GNL compromises some 105
but not all phenotypes when these deletion or mutant variants are 106
overexpressed in Arabidopsis and the effects of the overexpression 107
transgenes are compared to the effects of the overexpression of the wild type 108
proteins (Behringer et al 2014) The fact that the overexpression of every 109
LLM-domain B-GATA factor tested to date results in highly comparable 110
phenotypes suggested that LLM-domain B-GATAs have conserved 111
biochemical functions (Behringer et al 2014) 112
A range of studies have already addressed the upstream signaling pathways 113
controlling GNC and GNL expression GNC was initially identified as a nitrate-114
inducible gene with an apparent role in the control of greening (Bi et al 2005) 115
GNL was originally identified based on its strong CK- (cytokinin-)regulated 116
gene expression and therefore designated CYTOKININ-RESPONSIVE GATA 117
FACTOR1 (CGA1) (Naito et al 2007) The expression of GNC and GNL is 118
also controlled by the DELLA regulators of the GA (gibberellin) signaling 119
pathway as well as by the light-labile PIFs (PHYTOCHROME INTERACTING 120
FACTORS) of the phytochrome signaling pathway which are themselves 121
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
5
negatively regulated by DELLAs (Richter et al 2010 Leivar and Monte 122
2014) Furthermore the auxin signaling regulators ARF2 (AUXIN RESPONSE 123
FACTOR2) and ARF7 the flowering regulator SOC1 (SUPPRESSOR-OF-124
OVEREXPRESSION-OF-CONSTANS1) as well as the floral development 125
regulators AP3 (APETALA3) and (PI) PISTILLATA have been implicated in 126
GNC and GNL transcription control (Mara and Irish 2008 Richter et al 2010 127
Richter et al 2013 Richter et al 2013) Thus multiple signaling pathways 128
converge on GNC and GNL expression and the two genes control a broad 129
range of developmental responses in plants 130
To date nothing is known about the regulation and biological function of the 131
four Arabidopsis LLM-domain B-GATAs GATA15 GATA16 GATA17 and 132
GATA17L Here we examine the entire LLM-domain B-GATA gene family 133
through the analysis of single gene insertion mutants and a range of mutant 134
combinations These analyses reveal mainly overlapping roles for these B-135
GATAs in the control of previously known B-GATA-regulated processes such 136
as greening hypocotyl elongation flowering time and senescence but they 137
also reveal previously unknown functions of LLM-domain B-GATAs in 138
phyllotactic patterning floral organ initiation and accessory meristem 139
formation Several of these responses may be mediated by CK signaling and 140
CK treatments induce the expression of all six GATA genes CK-induced gene 141
expression is partially compromised in LLM-domain B-GATA mutants 142
suggesting that B-GATA genes play a role in CK responses Furthermore we 143
provide evidence for a cross-regulation of the different LLM-domain B-GATA 144
genes at the transcriptional level 145
146
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
6
RESULTS 147
Evolutionary conservation of the LLM-domain B-GATAs within the 148
Brassicaceae The Arabidopsis thaliana genome encodes six LLM-domain 149
GATA transcription factors namely GNC (AtGATA21) GNL (AtGATA22) as 150
well as GATA15 (AtGATA15) GATA16 (AtGATA16) GATA17 (AtGATA17) 151
and GATA17L (AtGATA17-LIKE) (Behringer et al 2014 Behringer and 152
Schwechheimer 2015) Phylogenetic analyses showed that these proteins 153
can be grouped into three pairs of highly homologous proteins comprised of 154
the with regard to the length of their N-termini long B-GATAs GNC and GNL 155
and the short B-GATAs GATA15 and GATA16 as well as GATA17 and 156
GATA17L respectively (Fig 1A and B Fig S1) (Behringer et al 2014 157
Behringer and Schwechheimer 2015) It had previously become apparent 158
that the respective subfamilies of long and short B-GATAs were conserved in 159
different plant species but that gene duplications in individual subfamilies may 160
have led to a differential expansion of these subfamilies in different species 161
(Behringer et al 2014) To gain an insight into the conservation of these 162
GATAs within the family of Brassicaceae we searched for LLM-domain B-163
GATAs in the fully sequenced genomes of Arabidopsis lyrata Capsella 164
rubella Eutrema salsugineum and Brassica rapa We retrieved orthologs for 165
each of the six Arabidopsis B-GATAs within each Brassicaceae species (Fig 166
S1 and Fig S2) Whereas the Arabidopsis lyrata Capsella rubella and 167
Eutrema salsugineum genomes contained exactly one apparent ortholog for 168
each of the six Arabidopsis thaliana genes the Brassica rapa genome known 169
to have undergone hexaploidization followed by genome fractionation 170
retained at least two orthologs each for GNC GNL GATA17 and GATA17L 171
and one ortholog of GATA15 and GATA16 (Fig S1 and Fig S2) (Wang et al 172
2011 Tang et al 2012) In summary we concluded that the six LLM-domain 173
B-GATAs are conserved in the different Brassicaceae examined here and that 174
four members of the gene family were present in multiple copies in Brassica 175
rapa 176
177
Characterization of insertion mutants of the six LLM-domain B-GATA 178
genes All previously published genetic analyses of the LLM-domain B-179
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
7
GATAs genes from Arabidopsis thaliana focused on GNC and GNL 180
(Behringer and Schwechheimer 2015) As yet no mutants have been 181
described for the remaining four LLM-domain B-GATAs and their biological 182
function remains elusive We therefore identified homozygous lines of T-DNA 183
insertion mutants for each of the six genes (Fig 1C Fig S3) and 184
subsequently analyzed these lines with regard to the effects of the respective 185
mutations on gene expression using quantitative real time PCR (qRT-PCR) 186
and semi-quantitative PCR (sqRT-PCR) (Fig S3B) The analysis of the gnc 187
allele (Salk_001778) which had already been used in several publications 188
suggested that neither the full-length transcript nor regions downstream from 189
the insertion site were transcribed (Fig 1C and Fig S3B) (Richter et al 2010) 190
(Bi et al 2005) Similarly analyses of the gnl insertion mutant (Salk_003995) 191
using primers spanning the entire transcript or flanking the T-DNA harbored in 192
the intron suggested that the GNL full-length transcript was absent (Fig 1C 193
and Fig S3B) (Bi et al 2005 Richter et al 2010) Although one of the 194
selected GATA15 insertion alleles (gata15-1 SAIL_618_B11) was annotated 195
as an in-gene insertion (wwwsignalsalkedu) we found no evidence for a 196
change of GATA15 gene expression in the homozygous insertion line (Fig 1C 197
and Fig S3B) Sequencing of the insertion site then revealed that the 198
insertion was downstream from the genersquos 3-UTR (untranslated region) 199
indicating that the GATA15 gene was intact in SAIL_618_B11 (Fig 1C and 200
Fig S3B) The analysis of a second allele (gata15-2 WiscDsLox471A10) with 201
an exon-insertion revealed a strong down-regulation of GATA15 expression in 202
both RT-PCR analyses (Fig 1C and Fig S3B) In a GATA16 allele with a T-203
DNA insertion in the genersquos 3-UTR (gata16-1 Salk_021471) transcript 204
abundance was partially reduced (Fig 1C and Fig S3B) The abundance of 205
GATA17 was also partially reduced in Salk_101994 (gata17-1) which 206
harbored a T-DNA insertion in the genes 5-UTR and strongly reduced in 207
SALK_049041 (gata17-2) which carried an insertion in the LLM-domain-208
encoding gene region (Fig 1C and Fig S3B) Finally we detected no 209
transcript in our analyses of a GATA17L exon insertion allele (gata17l-1 210
Salk_026798) when using primer pairs spanning the insertion or located 211
downstream from the insertion site (Fig 1C and Fig S3B) When analyzing 212
this allele with a primer combination upstream of the insertion we were able 213
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
8
to detect residual transcript in gata17l-1 but judged that this may not give rise 214
to a fully functional protein since the insertion was upstream of the LLM-215
domain (Fig 1C and Fig S3B) We concluded that the expression of the 216
respective GATA genes was absent or at least strongly reduced in alleles for 217
GNC (gnc) GNL (gnl) GATA15 (gata15-2) GATA17 (gata17-2) and 218
GATA17L (gata17l-1) alleles The gata16-1 and gata17-1 alleles were only 219
partially impaired in the expression of the respective genes whereas the 220
gata15-1 allele was not affected in GATA15 expression The gata15-2 and 221
gata17-2 alleles became available to us only later in this study and for 222
strategic reasons could not be included in the generation of the complex 223
mutant combinations described below Any of the phenotypes described in the 224
following sections for the more complex mutant combinations were not 225
apparent in the gata15-2 and gata17-2 single mutants We therefore exclude 226
the possibility that these genes are by themselves responsible for any of the 227
phenotypes described here for the higher order mutants 228
229
LLM-domain B-GATAs redundantly control greening We did not observe 230
any obvious defects in the gata single mutants apart from the already well-231
documented defects in greening of the gnc mutants (Bi et al 2005 Richter et 232
al 2010 Chiang et al 2012) To generate higher order mutants in the 233
closely related B-GATA genes we combined the alleles gnc gnl gata15-1 234
gata16-1 gata17-1 and gata17l-1 For simplicity we will subsequently omit 235
the respective allele numbers Since the presence of the misannotated 236
gata15-1 mutation does not provide information about the loss of GATA15 we 237
refer to this allele as GATA15 238
Through genetic crosses we obtained gata17 gata17l double mutants 239
GATA15 gata16 gata17 gata17l gnc gnl GATA15 gata16 and gnc gnl gata17 240
gata17l triple and quadruple mutants as well as a gnc gnl GATA15 gata16 241
gata17 gata17l quintuple mutant In the following we will refer to the gata 242
quintuple mutant as the quintuple mutant and to the GATA15 gata16 gata17 243
gata17l triple mutant that does not include the gnc and gnl alleles as the 244
(complementary) triple mutant In all other cases we will specify the allele 245
combinations 246
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
9
To examine a possible redundant function among the B-GATAs in the control 247
of greening we analyzed greening and chlorophyll content in 14 day-old light-248
grown gata mutant plants (Fig 1D and E) Although there was no indication 249
for a defect in greening in gata17 gata17l or in the triple mutant we found that 250
the greening defect of gnc gnl was further enhanced in the gnc gnl gata17 251
gata17l quadruple mutant and even more in the quintuple mutant (Fig 1D and 252
E) At the same time greening defects were not obvious in any of the gata 253
single mutants (Fig 1E) In the case of the gata15 and gata17 alleles this 254
was not due to the fact that weak alleles had been chosen for the complex 255
mutants since also the severe alleles gata15-2 and gata17-2 showed no 256
reduction in chlorophyll levels when compared to the wild type (Fig 1E) 257
The further reduction in chlorophyll accumulation in the quintuple mutant was 258
also apparent when we analyzed chlorophyll abundance by fluorescence 259
microscopy where we found reduced chloroplast fluorescence when 260
comparing the quintuple mutant to gnc gnl (Fig 1F) Furthermore we noted 261
that the greening defect gradually disappeared in gnc gnl mutants after the 262
seedling stage but remained apparent in the quintuple mutants also in adult 263
plants (Fig S4) Taken together this suggested that all mutated GATA genes 264
participate in the control of greening and chlorophyll accumulation and that 265
the defects of mutants defective in a single gene may be suppressed by the 266
presence of the other family members 267
268
Gene expression regulation of the LLM-domain B-GATAs GNC and GNL 269
had been in the focus of several studies based on the strong regulation of 270
their gene expression by light and CK (Bi et al 2005 Naito et al 2007 271
Richter et al 2010 Richter et al 2013) We were therefore interested in 272
evaluating to what extent light and CK regulate the expression of the other 273
GATA genes In the case of light regulation we found that far-red red as well 274
as blue light strongly induced the expression of GNL and to some extent also 275
the expression of GNC (Fig 2) In blue light we noted a subtle but significant 276
induction of GATA15 and GATA16 (Fig 2C) Importantly this regulation was 277
not observed in the red and far-red light receptor mutant phyA phyB or in the 278
blue light receptor mutant cry1 cry2 (Fig 2) Furthermore light-induced gene 279
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
10
expression changes in red light were impaired in the quadruple mutant for the 280
PHYTOCHROME INTERACTING FACTOR (PIF) genes PIF1 PIF3 PIF4 281
and PIF5 (pifq) indicating that the light-dependent regulation in red light was 282
mediated by the phytochrome and PIF signaling pathways (Fig S5) (Leivar et 283
al 2008) 284
The CK experiments indicated that CK induces the expression of all six GATA 285
genes (Fig 3) With an about three-fold increase in transcript abundance this 286
induction was strongest for GNL but could be observed for each of the other 287
GATA genes after a one to four hour CK treatment (Fig 3) Thus CK 288
promotes the expression of all six LLM-domain B-GATAs 289
290
Mutants of LLM-domain B-GATAs are impaired in hypocotyl elongation 291
Due to the strong regulation particularly of GNL but also of GNC in far-red red 292
and blue light conditions we examined the contribution of the GATA genes to 293
hypocotyl elongation by analyzing mutant seedlings in the different light 294
conditions in two different light intensities (Fig 4) Here we found that 295
quintuple mutant seedlings had a shorter hypocotyl than wild type seedlings in 296
white light as well as in red far-red and blue light (Fig 4) Notably these 297
phenotypes were not apparent or at least not significant in the gnc gnl 298
double mutant or the triple mutant suggesting that the respective other GATA 299
genes may contribute to the hypocotyl elongation phenotype in these 300
backgrounds (Fig 4) At the same time hypocotyl length of dark-grown 301
mutant seedlings was indistinguishable in all genotypes from that observed in 302
the wild type (Fig 4E and G) The short hypocotyl phenotype of the GATA 303
gene mutants was also interesting because we previously observed that the 304
LLM-domain B-GATA overexpressors had a longer hypocotyl than the wild 305
type when grown in white light (Behringer et al 2014) When analyzed in the 306
different light conditions we found the long hypocotyl phenotype of the GNL 307
overexpression line (GNLox) was also present in far-red and blue light but 308
interestingly not in red light where hypocotyls were even shorter than in the 309
wild type or the mutants when grown in weak red light conditions (Fig 4) 310
311
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
11
GATA gene mutants have differential gene expression defects The 312
strong induction of GNL gene expression by CK was already repeatedly 313
described and is the reason for the genersquos original designation CGA1 314
(CYTOKININ-INDUCED GATA1) (Naito et al 2007 Kollmer et al 2011) In 315
order to examine the role of the GATA genes in CK-responsive gene 316
expression we performed microarray experiments with 14 day-old wild type 317
plants gnc gnl and triple mutants as well as quintuple mutants treated for 60 318
min with the CK 6-BA (6-benzylaminopurine) or a corresponding mock solvent 319
control (Fig 5 and Fig 6 Supplemental Tables S1 and S2) We then 320
analyzed the resulting data with regard to differences in basal gene 321
expression (Fig 5) and with regard to gene expression differences after the 322
CK treatment in comparison to the mock control (Fig 6) For technical 323
reasons the experiment was conducted in two rounds the first experiment 324
included the Col-0 (1) wild type control the gnc gnl double mutant and the 325
quintuple mutant the second experiment included an independent Col-0 (2) 326
wild type control and the triple mutant When we examined the expression of 327
the CK-induced ARRs (A-TYPE RESPONSE REGULATORS) of the CK 328
signaling pathway we could confirm that the respective CK induction 329
experiments were comparably efficient between the two experiments and in 330
all genotypes tested (Fig S6A) We also examined the microarray dataset 331
with regard to the expression of a previously defined so-called ldquogolden listrdquo of 332
CK-regulated genes (Bhargava et al 2013) Over 70 of the 331 entities 333
derived from this list were significantly CK-regulated in our experiments 334
(Supplemental Table S3) When applying a two-fold expression change as 335
additional filter criterion 71 of these 331 entities were identified as up- (Col-0 336
1 and Col-0 2) and 39 (Col-0 1) and 25 entities (Col-0 2) as down-regulated 337
after CK treatment (Fig S6B and Supplemental Table S3) We thus 338
concluded that the CK treatments in both experiments were similarly efficient 339
and that the data sets could be used for comparative analyses 340
The comparison of the basal gene expression profiles of the wild type 341
samples and the mutants indicated that the (untreated) quintuple mutant 342
seedlings had by far the largest number of differentially regulated genes with 343
4722 down-regulated and 976 up-regulated entities when compared to the 344
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
12
Col-0 (1) wild type (Fig 5 and Supplemental Table S1) Furthermore the gnc 345
gnl double mutant contained 2458 down- and 294 up-regulated entities 346
respectively (Fig 5 and Supplemental Table S1) In comparison the number 347
of differentially expressed genes was comparatively minor in the triple mutant 348
which had only 323 down- and 278 up-regulated entities in this particular 349
experiment and selected physiological growth conditions (Fig 5) 350
The comparatively strong gene expression differences in the gnc gnl double 351
and the quintuple mutants and the fact that both mutants shared the gnc gnl 352
loss-of-function at the genetic level was reflected by the large overlap 353
between the differentially expressed gene sets of the two genotypes 354
Specifically 1922 of the 2458 down-regulated entities from gnc gnl were also 355
down-regulated in the quintuple mutant (Fig 5B) and 98 of the 294 entities 356
up-regulated in gnc gnl were also up-regulated in the quintuple mutant (Fig 357
5B) Conversely the overlap in gene expression defects between the triple 358
mutant and the quintuple mutant was higher than that between the triple 359
mutant and gnc gnl (Fig 5B) We therefore concluded that the gene 360
expression differences between the different GATA gene mutants was 361
strongest in the quintuple mutant and that the strong increase in the number 362
of differentially regulated genes exceeded by far the differences seen in the 363
gnc gnl double mutant and the triple mutant This supported our conclusion 364
from the phenotypic analyses that the GATA genes act in a functionally 365
redundant manner 366
Since we had observed increased gene expression of all six GATA genes 367
after CK-treatment we expected that CK-induced gene expression may be 368
compromised in the gata mutants (Fig 6) Indeed of the 996 entities that 369
were down-regulated after CK-treatment in the wild type (Col-0 1) only 173 370
(18 ) and 89 (9 ) remained CK-regulated in the quintuple and gnc gnl 371
mutant respectively (Fig 6A and Supplemental Table S2) At the same time 372
of the 188 entities that were down-regulated after CK treatment in the wild 373
type sample of the second experiment (Col-0 2) 83 (44 ) remained CK-374
regulated in the gata triple mutant (Fig 6B) The differential effect on gene 375
expression between the different mutants was less pronounced when 376
examining the CK-induced genes Here 49 (quintuple mutant) 59 (gnc 377
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
13
gnl) and 63 (triple mutant) of the CK-induced entities from the respective 378
wild types remained CK-regulated in the mutants (Fig 6A and B) In summary 379
we concluded that CK-regulated gene expression is impaired in the GATA 380
gene mutants 381
382
GATA gene mutants are defective in CK-regulated developmental 383
processes Based on the strong defects of gata mutants in CK-regulated 384
gene expression we searched for phenotypes that could be explained by 385
defects in CK response CK signaling was described as a regulator of 386
phyllotactic patterning in the shoot apical meristem of Arabidopsis (Besnard et 387
al 2014) In line with a role of the LLM-domain B-GATA genes in this process 388
we observed frequent aberrations from normal phyllotactic patterning in flower 389
positioning in triple and quintuple mutant inflorescences (Fig 7) Quantitative 390
analyses revealed aberrations from the canonical angle of ~1375deg in about 391
~30 of the petioles in triple and quintuple inflorescences (Fig 7D) At the 392
same time this patterning defect was not apparent in the gnc gnl double 393
mutant Since we also did not observe a quantitative increase in the severity 394
of this phenotype between the triple and the quintuple mutant we concluded 395
that GATA16 GATA17 and GATA17L may be responsible for this phenotype 396
(Fig 7D) 397
Leaf senescence is another CK-regulated process (Hwang et al 2012) In the 398
wild type CK levels are reduced during senescence and positive or negative 399
interference with the decrease in CK levels can promote and delay 400
senescence respectively (Hwang et al 2012) Senescence can be induced 401
by shifting light-grown plants to the dark and we had previously already shown 402
that leaf senescence is delayed in overexpression lines of LLM-domain B-403
GATAs (Behringer et al 2014) To examine possible senescence defects in 404
the gata mutants we floated leaves of wild type and mutant plants in the dark 405
on a mock solution or on a solution containing 005 microM or 01 microM 6-BA (Fig 406
8A ndash F) (Weaver and Amasino 2001) In line with the expectation that the 407
presence of CK in the medium would suppress the senescence process we 408
observed decreased senescence in CK-treated wild type leaves which we 409
quantitatively assessed by measuring chlorophyll levels in the mock- and CK-410
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
14
treated leaves (Fig 8F) Here we found that the effects of the senescence 411
treatment were less efficiently suppressed by CK treatments in the gnc gnl 412
double and in the quintuple mutant than in the wild type or the triple mutant 413
(Fig 8A - F) Similarly we observed a stronger effect on the decreases in 414
chlorophyll and total protein levels in the gnc gnl as well as the quintuple 415
mutant when we transferred light-grown plants for four days to the dark (Fig 416
8G ndash I) As previously reported GNL overexpressing plants (GNLox) had 417
senescence phenotypes that were antagonistic to the ones observed with the 418
mutants (Fig 8) (Behringer et al 2014) In the case of the leaf floating assay 419
CK-treatment had a stronger effect on greening and chlorophyll abundance in 420
GNLox than in the wild type (Fig 8A ndash F) When we transferred plants from 421
the light to the dark GNLox seedlings remained visibly green and did not 422
display the strong decrease in total protein abundance that we observed in 423
the wild type or the GATA gene mutants (Fig 8G ndash I) We thus concluded that 424
GATA gene mutants and overexpressors have antagonistic effects with 425
regard to the regulation of senescence and that GNC and GNL contribute 426
particularly strongly to this phenotype 427
CK induces greening in light-grown seedlings (Kobayashi et al 2012) To 428
examine to what extent CK can induce chlorophyll accumulation in the gata 429
mutants we grew plants for two weeks in constant light on medium containing 430
CK (5 nM BA) or a corresponding CK-free medium As expected chlorophyll 431
content was increased in wild type plants but this increase was neither 432
significant in the gnc gnl nor the triple or the quintuple mutants (Fig S7) We 433
thus concluded that the GATA factors are required for CK-induced greening in 434
Arabidopsis seedlings 435
It was previously reported that hypocotyl elongation could be repressed by 436
incubating dark-grown seedlings in the presence of CK (Su and Howell 1995) 437
Since the GATA gene mutants had displayed a hypocotyl elongation defect in 438
light-grown seedlings (Fig 4) we also explored the link between hypocotyl 439
elongation and CK However when we tested the effects of CK on hypocotyl 440
elongation in dark-grown seedlings we did not detect any significant 441
differences in the response between the different genotypes tested (Fig S8) 442
It is thus unlikely that the GATA genes participate in this elongation response 443
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
15
Branching is another phenotype that has been attributed to defects in CK 444
biosynthesis and response (El-Showk et al 2013) When we analyzed the 445
general habitus of the quintuple mutants we noted that they had an increased 446
number of secondary branches that was combined with a reduced plant 447
height (Fig 9A - C) Occasionally we also observed the outgrowth of 448
inflorescences in the axils of lateral inflorescences in the mutants whereas we 449
had not noted this phenotype in the wild type (Fig 9D and E) A quantitative 450
analysis of these phenotypes over all gata mutant phenotypes revealed that 451
the number of first and second order lateral branches increased with 452
increasing mutant complexity (Fig 9B) Here we observed strong effects 453
already in the gata17 gata17l mutant as well as the gnc gnl double mutant but 454
strongest defects in the triple and the quintuple mutant thus all five GATA 455
genes may contribute to this phenotype (Fig 9B) At the same time plant 456
height was reduced to a significant but still minor level in these mutants 457
suggesting that decreases in plant height which may have indirect 458
consequences on the general plant habitus and branching are not the 459
primary cause for altered branching patterns in the gata mutants (Fig 9C) It 460
is further important to note that the higher order GATA gene mutants initiated 461
fewer vegetative buds but had a higher number of growing buds as reflected 462
by the increased branch numbers (Fig 9F) Thus axillary bud formation is 463
promoted and bud outgrowth is repressed by the GATAs in the wild type 464
Another phenotype that became apparent during our analysis of the gata 465
mutant plant habitus was an increase in the angle formed between the 466
primary inflorescence and the lateral inflorescences (Fig 9G and H Fig S9) 467
gata mutants had a decreased angle formed between the primary 468
inflorescence and lateral inflorescences and this phenotype was strongest in 469
the quintuple mutant and intermediate in the gnc gnl and triple mutants (Fig 470
9G and H Fig S9) Interestingly we had previously observed increased 471
branch angles in GATA overexpression lines such as GNLox (Fig S9) 472
(Behringer et al 2014) We thus concluded that GATA gene mutants and 473
overexpressors antagonistically regulate branch angles in Arabidopsis 474
475
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
3
INTRODUCTION 57
Transcription factors of the GATA family contain a type IV zinc finger DNA-58
binding domain (C-X2-C-X17-20-C-X2-C) which is predicted to bind the 59
consensus DNA sequence WGATAR (where W is T or A and R is G or A) 60
(Reyes et al 2004) The genomes of higher plants encode approximately 30 61
GATA transcription factors which are classified as A- B- C- and D-GATAs 62
according to their primary amino acid sequence the conservation of their 63
GATA DNA-binding domains the conservation of protein domains outside of 64
the GATA DNA-binding domain and their exon-intron structures (Reyes et al 65
2004) We recently showed that the Arabidopsis thaliana B-GATAs can be 66
further subdivided into two structural subfamilies with either a HAN- or an 67
LLM-domain located N- or C-terminally of the GATA DNA-binding domain 68
(Behringer et al 2014 Behringer and Schwechheimer 2015) The HAN-69
domain was initially identified as a conserved domain in the floral morphology 70
regulator HAN (HANABA TARANU) (Zhao et al 2004 Nawy et al 2010 71
Zhang et al 2013) the LLM-domain was named after the invariant leucine-72
leucine-methionine (LLM) motif in the conserved C-terminus of the respective 73
B-GATAs (Richter et al 2010 Behringer et al 2014) 74
A recent comparative analysis of B-GATAs from Arabidopsis Solanum 75
lycopersicon (tomato) Hordeum vulgare (barley) and Brachipodium 76
distachyon (Brachipodium) revealed that HAN-domain and LLM-domain B-77
GATAs have different biological and biochemical activities the expression of 78
HAN-domain B-GATAs under the control of an LLM-domain B-GATA 79
promoter could not complement LLM-domain B-GATA loss-of-function 80
mutants and the overexpression of HAN- and LLM-domain B-GATAs in 81
Arabidopsis differentially affected plant growth (Behringer et al 2014 82
Behringer and Schwechheimer 2015) The existence of the Brassicaceae-83
specific GATA23 which has a degenerate LLM-domain as well as the 84
existence of monocot-specific HAN-domain B-GATAs reveal that expansions 85
of the B-GATA family have been used during plant evolution to control specific 86
aspects of plant development (De Rybel et al 2010 Whipple et al 2010 87
Behringer and Schwechheimer 2015) 88
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
4
The Arabidopsis LLM-domain B-GATA family is comprised of GATA15 89
GATA16 GATA17 and GATA17L (GATA17-LIKE) as well as GNC (GATA 90
NITRATE-INDUCIBLE CARBON METABOLISM-INVOLVED GATA21 91
hitherto GNC) and GNL (GNC-LIKECYTOKININ-RESPONSIVE GATA 92
FACTOR1 hitherto GNL) (Behringer et al 2014 Behringer and 93
Schwechheimer 2015) Among these the paralogous GNC and GNL are 94
already well characterized with regard to their biological function and their 95
upstream regulation gnc and gnc gnl mutants are defective in greening and 96
chloroplast biogenesis and they germinate and flower slightly earlier than the 97
wild type (Bi et al 2005 Richter et al 2010 Chiang et al 2012 Richter et 98
al 2013) Conversely overexpression of the two GATAs results in a strong 99
increase in chlorophyll accumulation and delayed germination and flowering 100
(Richter et al 2010 Richter et al 2013 Behringer et al 2014) In addition 101
GNC and GNL overexpression promotes hypocotyl elongation in light-grown 102
seedlings alterations in leaf shape and an increased angle between the 103
primary and lateral inflorescences (Behringer et al 2014) Importantly 104
deletion or mutation of the LLM-domain of GNC or GNL compromises some 105
but not all phenotypes when these deletion or mutant variants are 106
overexpressed in Arabidopsis and the effects of the overexpression 107
transgenes are compared to the effects of the overexpression of the wild type 108
proteins (Behringer et al 2014) The fact that the overexpression of every 109
LLM-domain B-GATA factor tested to date results in highly comparable 110
phenotypes suggested that LLM-domain B-GATAs have conserved 111
biochemical functions (Behringer et al 2014) 112
A range of studies have already addressed the upstream signaling pathways 113
controlling GNC and GNL expression GNC was initially identified as a nitrate-114
inducible gene with an apparent role in the control of greening (Bi et al 2005) 115
GNL was originally identified based on its strong CK- (cytokinin-)regulated 116
gene expression and therefore designated CYTOKININ-RESPONSIVE GATA 117
FACTOR1 (CGA1) (Naito et al 2007) The expression of GNC and GNL is 118
also controlled by the DELLA regulators of the GA (gibberellin) signaling 119
pathway as well as by the light-labile PIFs (PHYTOCHROME INTERACTING 120
FACTORS) of the phytochrome signaling pathway which are themselves 121
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
5
negatively regulated by DELLAs (Richter et al 2010 Leivar and Monte 122
2014) Furthermore the auxin signaling regulators ARF2 (AUXIN RESPONSE 123
FACTOR2) and ARF7 the flowering regulator SOC1 (SUPPRESSOR-OF-124
OVEREXPRESSION-OF-CONSTANS1) as well as the floral development 125
regulators AP3 (APETALA3) and (PI) PISTILLATA have been implicated in 126
GNC and GNL transcription control (Mara and Irish 2008 Richter et al 2010 127
Richter et al 2013 Richter et al 2013) Thus multiple signaling pathways 128
converge on GNC and GNL expression and the two genes control a broad 129
range of developmental responses in plants 130
To date nothing is known about the regulation and biological function of the 131
four Arabidopsis LLM-domain B-GATAs GATA15 GATA16 GATA17 and 132
GATA17L Here we examine the entire LLM-domain B-GATA gene family 133
through the analysis of single gene insertion mutants and a range of mutant 134
combinations These analyses reveal mainly overlapping roles for these B-135
GATAs in the control of previously known B-GATA-regulated processes such 136
as greening hypocotyl elongation flowering time and senescence but they 137
also reveal previously unknown functions of LLM-domain B-GATAs in 138
phyllotactic patterning floral organ initiation and accessory meristem 139
formation Several of these responses may be mediated by CK signaling and 140
CK treatments induce the expression of all six GATA genes CK-induced gene 141
expression is partially compromised in LLM-domain B-GATA mutants 142
suggesting that B-GATA genes play a role in CK responses Furthermore we 143
provide evidence for a cross-regulation of the different LLM-domain B-GATA 144
genes at the transcriptional level 145
146
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
6
RESULTS 147
Evolutionary conservation of the LLM-domain B-GATAs within the 148
Brassicaceae The Arabidopsis thaliana genome encodes six LLM-domain 149
GATA transcription factors namely GNC (AtGATA21) GNL (AtGATA22) as 150
well as GATA15 (AtGATA15) GATA16 (AtGATA16) GATA17 (AtGATA17) 151
and GATA17L (AtGATA17-LIKE) (Behringer et al 2014 Behringer and 152
Schwechheimer 2015) Phylogenetic analyses showed that these proteins 153
can be grouped into three pairs of highly homologous proteins comprised of 154
the with regard to the length of their N-termini long B-GATAs GNC and GNL 155
and the short B-GATAs GATA15 and GATA16 as well as GATA17 and 156
GATA17L respectively (Fig 1A and B Fig S1) (Behringer et al 2014 157
Behringer and Schwechheimer 2015) It had previously become apparent 158
that the respective subfamilies of long and short B-GATAs were conserved in 159
different plant species but that gene duplications in individual subfamilies may 160
have led to a differential expansion of these subfamilies in different species 161
(Behringer et al 2014) To gain an insight into the conservation of these 162
GATAs within the family of Brassicaceae we searched for LLM-domain B-163
GATAs in the fully sequenced genomes of Arabidopsis lyrata Capsella 164
rubella Eutrema salsugineum and Brassica rapa We retrieved orthologs for 165
each of the six Arabidopsis B-GATAs within each Brassicaceae species (Fig 166
S1 and Fig S2) Whereas the Arabidopsis lyrata Capsella rubella and 167
Eutrema salsugineum genomes contained exactly one apparent ortholog for 168
each of the six Arabidopsis thaliana genes the Brassica rapa genome known 169
to have undergone hexaploidization followed by genome fractionation 170
retained at least two orthologs each for GNC GNL GATA17 and GATA17L 171
and one ortholog of GATA15 and GATA16 (Fig S1 and Fig S2) (Wang et al 172
2011 Tang et al 2012) In summary we concluded that the six LLM-domain 173
B-GATAs are conserved in the different Brassicaceae examined here and that 174
four members of the gene family were present in multiple copies in Brassica 175
rapa 176
177
Characterization of insertion mutants of the six LLM-domain B-GATA 178
genes All previously published genetic analyses of the LLM-domain B-179
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
7
GATAs genes from Arabidopsis thaliana focused on GNC and GNL 180
(Behringer and Schwechheimer 2015) As yet no mutants have been 181
described for the remaining four LLM-domain B-GATAs and their biological 182
function remains elusive We therefore identified homozygous lines of T-DNA 183
insertion mutants for each of the six genes (Fig 1C Fig S3) and 184
subsequently analyzed these lines with regard to the effects of the respective 185
mutations on gene expression using quantitative real time PCR (qRT-PCR) 186
and semi-quantitative PCR (sqRT-PCR) (Fig S3B) The analysis of the gnc 187
allele (Salk_001778) which had already been used in several publications 188
suggested that neither the full-length transcript nor regions downstream from 189
the insertion site were transcribed (Fig 1C and Fig S3B) (Richter et al 2010) 190
(Bi et al 2005) Similarly analyses of the gnl insertion mutant (Salk_003995) 191
using primers spanning the entire transcript or flanking the T-DNA harbored in 192
the intron suggested that the GNL full-length transcript was absent (Fig 1C 193
and Fig S3B) (Bi et al 2005 Richter et al 2010) Although one of the 194
selected GATA15 insertion alleles (gata15-1 SAIL_618_B11) was annotated 195
as an in-gene insertion (wwwsignalsalkedu) we found no evidence for a 196
change of GATA15 gene expression in the homozygous insertion line (Fig 1C 197
and Fig S3B) Sequencing of the insertion site then revealed that the 198
insertion was downstream from the genersquos 3-UTR (untranslated region) 199
indicating that the GATA15 gene was intact in SAIL_618_B11 (Fig 1C and 200
Fig S3B) The analysis of a second allele (gata15-2 WiscDsLox471A10) with 201
an exon-insertion revealed a strong down-regulation of GATA15 expression in 202
both RT-PCR analyses (Fig 1C and Fig S3B) In a GATA16 allele with a T-203
DNA insertion in the genersquos 3-UTR (gata16-1 Salk_021471) transcript 204
abundance was partially reduced (Fig 1C and Fig S3B) The abundance of 205
GATA17 was also partially reduced in Salk_101994 (gata17-1) which 206
harbored a T-DNA insertion in the genes 5-UTR and strongly reduced in 207
SALK_049041 (gata17-2) which carried an insertion in the LLM-domain-208
encoding gene region (Fig 1C and Fig S3B) Finally we detected no 209
transcript in our analyses of a GATA17L exon insertion allele (gata17l-1 210
Salk_026798) when using primer pairs spanning the insertion or located 211
downstream from the insertion site (Fig 1C and Fig S3B) When analyzing 212
this allele with a primer combination upstream of the insertion we were able 213
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
8
to detect residual transcript in gata17l-1 but judged that this may not give rise 214
to a fully functional protein since the insertion was upstream of the LLM-215
domain (Fig 1C and Fig S3B) We concluded that the expression of the 216
respective GATA genes was absent or at least strongly reduced in alleles for 217
GNC (gnc) GNL (gnl) GATA15 (gata15-2) GATA17 (gata17-2) and 218
GATA17L (gata17l-1) alleles The gata16-1 and gata17-1 alleles were only 219
partially impaired in the expression of the respective genes whereas the 220
gata15-1 allele was not affected in GATA15 expression The gata15-2 and 221
gata17-2 alleles became available to us only later in this study and for 222
strategic reasons could not be included in the generation of the complex 223
mutant combinations described below Any of the phenotypes described in the 224
following sections for the more complex mutant combinations were not 225
apparent in the gata15-2 and gata17-2 single mutants We therefore exclude 226
the possibility that these genes are by themselves responsible for any of the 227
phenotypes described here for the higher order mutants 228
229
LLM-domain B-GATAs redundantly control greening We did not observe 230
any obvious defects in the gata single mutants apart from the already well-231
documented defects in greening of the gnc mutants (Bi et al 2005 Richter et 232
al 2010 Chiang et al 2012) To generate higher order mutants in the 233
closely related B-GATA genes we combined the alleles gnc gnl gata15-1 234
gata16-1 gata17-1 and gata17l-1 For simplicity we will subsequently omit 235
the respective allele numbers Since the presence of the misannotated 236
gata15-1 mutation does not provide information about the loss of GATA15 we 237
refer to this allele as GATA15 238
Through genetic crosses we obtained gata17 gata17l double mutants 239
GATA15 gata16 gata17 gata17l gnc gnl GATA15 gata16 and gnc gnl gata17 240
gata17l triple and quadruple mutants as well as a gnc gnl GATA15 gata16 241
gata17 gata17l quintuple mutant In the following we will refer to the gata 242
quintuple mutant as the quintuple mutant and to the GATA15 gata16 gata17 243
gata17l triple mutant that does not include the gnc and gnl alleles as the 244
(complementary) triple mutant In all other cases we will specify the allele 245
combinations 246
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
9
To examine a possible redundant function among the B-GATAs in the control 247
of greening we analyzed greening and chlorophyll content in 14 day-old light-248
grown gata mutant plants (Fig 1D and E) Although there was no indication 249
for a defect in greening in gata17 gata17l or in the triple mutant we found that 250
the greening defect of gnc gnl was further enhanced in the gnc gnl gata17 251
gata17l quadruple mutant and even more in the quintuple mutant (Fig 1D and 252
E) At the same time greening defects were not obvious in any of the gata 253
single mutants (Fig 1E) In the case of the gata15 and gata17 alleles this 254
was not due to the fact that weak alleles had been chosen for the complex 255
mutants since also the severe alleles gata15-2 and gata17-2 showed no 256
reduction in chlorophyll levels when compared to the wild type (Fig 1E) 257
The further reduction in chlorophyll accumulation in the quintuple mutant was 258
also apparent when we analyzed chlorophyll abundance by fluorescence 259
microscopy where we found reduced chloroplast fluorescence when 260
comparing the quintuple mutant to gnc gnl (Fig 1F) Furthermore we noted 261
that the greening defect gradually disappeared in gnc gnl mutants after the 262
seedling stage but remained apparent in the quintuple mutants also in adult 263
plants (Fig S4) Taken together this suggested that all mutated GATA genes 264
participate in the control of greening and chlorophyll accumulation and that 265
the defects of mutants defective in a single gene may be suppressed by the 266
presence of the other family members 267
268
Gene expression regulation of the LLM-domain B-GATAs GNC and GNL 269
had been in the focus of several studies based on the strong regulation of 270
their gene expression by light and CK (Bi et al 2005 Naito et al 2007 271
Richter et al 2010 Richter et al 2013) We were therefore interested in 272
evaluating to what extent light and CK regulate the expression of the other 273
GATA genes In the case of light regulation we found that far-red red as well 274
as blue light strongly induced the expression of GNL and to some extent also 275
the expression of GNC (Fig 2) In blue light we noted a subtle but significant 276
induction of GATA15 and GATA16 (Fig 2C) Importantly this regulation was 277
not observed in the red and far-red light receptor mutant phyA phyB or in the 278
blue light receptor mutant cry1 cry2 (Fig 2) Furthermore light-induced gene 279
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
10
expression changes in red light were impaired in the quadruple mutant for the 280
PHYTOCHROME INTERACTING FACTOR (PIF) genes PIF1 PIF3 PIF4 281
and PIF5 (pifq) indicating that the light-dependent regulation in red light was 282
mediated by the phytochrome and PIF signaling pathways (Fig S5) (Leivar et 283
al 2008) 284
The CK experiments indicated that CK induces the expression of all six GATA 285
genes (Fig 3) With an about three-fold increase in transcript abundance this 286
induction was strongest for GNL but could be observed for each of the other 287
GATA genes after a one to four hour CK treatment (Fig 3) Thus CK 288
promotes the expression of all six LLM-domain B-GATAs 289
290
Mutants of LLM-domain B-GATAs are impaired in hypocotyl elongation 291
Due to the strong regulation particularly of GNL but also of GNC in far-red red 292
and blue light conditions we examined the contribution of the GATA genes to 293
hypocotyl elongation by analyzing mutant seedlings in the different light 294
conditions in two different light intensities (Fig 4) Here we found that 295
quintuple mutant seedlings had a shorter hypocotyl than wild type seedlings in 296
white light as well as in red far-red and blue light (Fig 4) Notably these 297
phenotypes were not apparent or at least not significant in the gnc gnl 298
double mutant or the triple mutant suggesting that the respective other GATA 299
genes may contribute to the hypocotyl elongation phenotype in these 300
backgrounds (Fig 4) At the same time hypocotyl length of dark-grown 301
mutant seedlings was indistinguishable in all genotypes from that observed in 302
the wild type (Fig 4E and G) The short hypocotyl phenotype of the GATA 303
gene mutants was also interesting because we previously observed that the 304
LLM-domain B-GATA overexpressors had a longer hypocotyl than the wild 305
type when grown in white light (Behringer et al 2014) When analyzed in the 306
different light conditions we found the long hypocotyl phenotype of the GNL 307
overexpression line (GNLox) was also present in far-red and blue light but 308
interestingly not in red light where hypocotyls were even shorter than in the 309
wild type or the mutants when grown in weak red light conditions (Fig 4) 310
311
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
11
GATA gene mutants have differential gene expression defects The 312
strong induction of GNL gene expression by CK was already repeatedly 313
described and is the reason for the genersquos original designation CGA1 314
(CYTOKININ-INDUCED GATA1) (Naito et al 2007 Kollmer et al 2011) In 315
order to examine the role of the GATA genes in CK-responsive gene 316
expression we performed microarray experiments with 14 day-old wild type 317
plants gnc gnl and triple mutants as well as quintuple mutants treated for 60 318
min with the CK 6-BA (6-benzylaminopurine) or a corresponding mock solvent 319
control (Fig 5 and Fig 6 Supplemental Tables S1 and S2) We then 320
analyzed the resulting data with regard to differences in basal gene 321
expression (Fig 5) and with regard to gene expression differences after the 322
CK treatment in comparison to the mock control (Fig 6) For technical 323
reasons the experiment was conducted in two rounds the first experiment 324
included the Col-0 (1) wild type control the gnc gnl double mutant and the 325
quintuple mutant the second experiment included an independent Col-0 (2) 326
wild type control and the triple mutant When we examined the expression of 327
the CK-induced ARRs (A-TYPE RESPONSE REGULATORS) of the CK 328
signaling pathway we could confirm that the respective CK induction 329
experiments were comparably efficient between the two experiments and in 330
all genotypes tested (Fig S6A) We also examined the microarray dataset 331
with regard to the expression of a previously defined so-called ldquogolden listrdquo of 332
CK-regulated genes (Bhargava et al 2013) Over 70 of the 331 entities 333
derived from this list were significantly CK-regulated in our experiments 334
(Supplemental Table S3) When applying a two-fold expression change as 335
additional filter criterion 71 of these 331 entities were identified as up- (Col-0 336
1 and Col-0 2) and 39 (Col-0 1) and 25 entities (Col-0 2) as down-regulated 337
after CK treatment (Fig S6B and Supplemental Table S3) We thus 338
concluded that the CK treatments in both experiments were similarly efficient 339
and that the data sets could be used for comparative analyses 340
The comparison of the basal gene expression profiles of the wild type 341
samples and the mutants indicated that the (untreated) quintuple mutant 342
seedlings had by far the largest number of differentially regulated genes with 343
4722 down-regulated and 976 up-regulated entities when compared to the 344
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
12
Col-0 (1) wild type (Fig 5 and Supplemental Table S1) Furthermore the gnc 345
gnl double mutant contained 2458 down- and 294 up-regulated entities 346
respectively (Fig 5 and Supplemental Table S1) In comparison the number 347
of differentially expressed genes was comparatively minor in the triple mutant 348
which had only 323 down- and 278 up-regulated entities in this particular 349
experiment and selected physiological growth conditions (Fig 5) 350
The comparatively strong gene expression differences in the gnc gnl double 351
and the quintuple mutants and the fact that both mutants shared the gnc gnl 352
loss-of-function at the genetic level was reflected by the large overlap 353
between the differentially expressed gene sets of the two genotypes 354
Specifically 1922 of the 2458 down-regulated entities from gnc gnl were also 355
down-regulated in the quintuple mutant (Fig 5B) and 98 of the 294 entities 356
up-regulated in gnc gnl were also up-regulated in the quintuple mutant (Fig 357
5B) Conversely the overlap in gene expression defects between the triple 358
mutant and the quintuple mutant was higher than that between the triple 359
mutant and gnc gnl (Fig 5B) We therefore concluded that the gene 360
expression differences between the different GATA gene mutants was 361
strongest in the quintuple mutant and that the strong increase in the number 362
of differentially regulated genes exceeded by far the differences seen in the 363
gnc gnl double mutant and the triple mutant This supported our conclusion 364
from the phenotypic analyses that the GATA genes act in a functionally 365
redundant manner 366
Since we had observed increased gene expression of all six GATA genes 367
after CK-treatment we expected that CK-induced gene expression may be 368
compromised in the gata mutants (Fig 6) Indeed of the 996 entities that 369
were down-regulated after CK-treatment in the wild type (Col-0 1) only 173 370
(18 ) and 89 (9 ) remained CK-regulated in the quintuple and gnc gnl 371
mutant respectively (Fig 6A and Supplemental Table S2) At the same time 372
of the 188 entities that were down-regulated after CK treatment in the wild 373
type sample of the second experiment (Col-0 2) 83 (44 ) remained CK-374
regulated in the gata triple mutant (Fig 6B) The differential effect on gene 375
expression between the different mutants was less pronounced when 376
examining the CK-induced genes Here 49 (quintuple mutant) 59 (gnc 377
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
13
gnl) and 63 (triple mutant) of the CK-induced entities from the respective 378
wild types remained CK-regulated in the mutants (Fig 6A and B) In summary 379
we concluded that CK-regulated gene expression is impaired in the GATA 380
gene mutants 381
382
GATA gene mutants are defective in CK-regulated developmental 383
processes Based on the strong defects of gata mutants in CK-regulated 384
gene expression we searched for phenotypes that could be explained by 385
defects in CK response CK signaling was described as a regulator of 386
phyllotactic patterning in the shoot apical meristem of Arabidopsis (Besnard et 387
al 2014) In line with a role of the LLM-domain B-GATA genes in this process 388
we observed frequent aberrations from normal phyllotactic patterning in flower 389
positioning in triple and quintuple mutant inflorescences (Fig 7) Quantitative 390
analyses revealed aberrations from the canonical angle of ~1375deg in about 391
~30 of the petioles in triple and quintuple inflorescences (Fig 7D) At the 392
same time this patterning defect was not apparent in the gnc gnl double 393
mutant Since we also did not observe a quantitative increase in the severity 394
of this phenotype between the triple and the quintuple mutant we concluded 395
that GATA16 GATA17 and GATA17L may be responsible for this phenotype 396
(Fig 7D) 397
Leaf senescence is another CK-regulated process (Hwang et al 2012) In the 398
wild type CK levels are reduced during senescence and positive or negative 399
interference with the decrease in CK levels can promote and delay 400
senescence respectively (Hwang et al 2012) Senescence can be induced 401
by shifting light-grown plants to the dark and we had previously already shown 402
that leaf senescence is delayed in overexpression lines of LLM-domain B-403
GATAs (Behringer et al 2014) To examine possible senescence defects in 404
the gata mutants we floated leaves of wild type and mutant plants in the dark 405
on a mock solution or on a solution containing 005 microM or 01 microM 6-BA (Fig 406
8A ndash F) (Weaver and Amasino 2001) In line with the expectation that the 407
presence of CK in the medium would suppress the senescence process we 408
observed decreased senescence in CK-treated wild type leaves which we 409
quantitatively assessed by measuring chlorophyll levels in the mock- and CK-410
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
14
treated leaves (Fig 8F) Here we found that the effects of the senescence 411
treatment were less efficiently suppressed by CK treatments in the gnc gnl 412
double and in the quintuple mutant than in the wild type or the triple mutant 413
(Fig 8A - F) Similarly we observed a stronger effect on the decreases in 414
chlorophyll and total protein levels in the gnc gnl as well as the quintuple 415
mutant when we transferred light-grown plants for four days to the dark (Fig 416
8G ndash I) As previously reported GNL overexpressing plants (GNLox) had 417
senescence phenotypes that were antagonistic to the ones observed with the 418
mutants (Fig 8) (Behringer et al 2014) In the case of the leaf floating assay 419
CK-treatment had a stronger effect on greening and chlorophyll abundance in 420
GNLox than in the wild type (Fig 8A ndash F) When we transferred plants from 421
the light to the dark GNLox seedlings remained visibly green and did not 422
display the strong decrease in total protein abundance that we observed in 423
the wild type or the GATA gene mutants (Fig 8G ndash I) We thus concluded that 424
GATA gene mutants and overexpressors have antagonistic effects with 425
regard to the regulation of senescence and that GNC and GNL contribute 426
particularly strongly to this phenotype 427
CK induces greening in light-grown seedlings (Kobayashi et al 2012) To 428
examine to what extent CK can induce chlorophyll accumulation in the gata 429
mutants we grew plants for two weeks in constant light on medium containing 430
CK (5 nM BA) or a corresponding CK-free medium As expected chlorophyll 431
content was increased in wild type plants but this increase was neither 432
significant in the gnc gnl nor the triple or the quintuple mutants (Fig S7) We 433
thus concluded that the GATA factors are required for CK-induced greening in 434
Arabidopsis seedlings 435
It was previously reported that hypocotyl elongation could be repressed by 436
incubating dark-grown seedlings in the presence of CK (Su and Howell 1995) 437
Since the GATA gene mutants had displayed a hypocotyl elongation defect in 438
light-grown seedlings (Fig 4) we also explored the link between hypocotyl 439
elongation and CK However when we tested the effects of CK on hypocotyl 440
elongation in dark-grown seedlings we did not detect any significant 441
differences in the response between the different genotypes tested (Fig S8) 442
It is thus unlikely that the GATA genes participate in this elongation response 443
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
15
Branching is another phenotype that has been attributed to defects in CK 444
biosynthesis and response (El-Showk et al 2013) When we analyzed the 445
general habitus of the quintuple mutants we noted that they had an increased 446
number of secondary branches that was combined with a reduced plant 447
height (Fig 9A - C) Occasionally we also observed the outgrowth of 448
inflorescences in the axils of lateral inflorescences in the mutants whereas we 449
had not noted this phenotype in the wild type (Fig 9D and E) A quantitative 450
analysis of these phenotypes over all gata mutant phenotypes revealed that 451
the number of first and second order lateral branches increased with 452
increasing mutant complexity (Fig 9B) Here we observed strong effects 453
already in the gata17 gata17l mutant as well as the gnc gnl double mutant but 454
strongest defects in the triple and the quintuple mutant thus all five GATA 455
genes may contribute to this phenotype (Fig 9B) At the same time plant 456
height was reduced to a significant but still minor level in these mutants 457
suggesting that decreases in plant height which may have indirect 458
consequences on the general plant habitus and branching are not the 459
primary cause for altered branching patterns in the gata mutants (Fig 9C) It 460
is further important to note that the higher order GATA gene mutants initiated 461
fewer vegetative buds but had a higher number of growing buds as reflected 462
by the increased branch numbers (Fig 9F) Thus axillary bud formation is 463
promoted and bud outgrowth is repressed by the GATAs in the wild type 464
Another phenotype that became apparent during our analysis of the gata 465
mutant plant habitus was an increase in the angle formed between the 466
primary inflorescence and the lateral inflorescences (Fig 9G and H Fig S9) 467
gata mutants had a decreased angle formed between the primary 468
inflorescence and lateral inflorescences and this phenotype was strongest in 469
the quintuple mutant and intermediate in the gnc gnl and triple mutants (Fig 470
9G and H Fig S9) Interestingly we had previously observed increased 471
branch angles in GATA overexpression lines such as GNLox (Fig S9) 472
(Behringer et al 2014) We thus concluded that GATA gene mutants and 473
overexpressors antagonistically regulate branch angles in Arabidopsis 474
475
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
4
The Arabidopsis LLM-domain B-GATA family is comprised of GATA15 89
GATA16 GATA17 and GATA17L (GATA17-LIKE) as well as GNC (GATA 90
NITRATE-INDUCIBLE CARBON METABOLISM-INVOLVED GATA21 91
hitherto GNC) and GNL (GNC-LIKECYTOKININ-RESPONSIVE GATA 92
FACTOR1 hitherto GNL) (Behringer et al 2014 Behringer and 93
Schwechheimer 2015) Among these the paralogous GNC and GNL are 94
already well characterized with regard to their biological function and their 95
upstream regulation gnc and gnc gnl mutants are defective in greening and 96
chloroplast biogenesis and they germinate and flower slightly earlier than the 97
wild type (Bi et al 2005 Richter et al 2010 Chiang et al 2012 Richter et 98
al 2013) Conversely overexpression of the two GATAs results in a strong 99
increase in chlorophyll accumulation and delayed germination and flowering 100
(Richter et al 2010 Richter et al 2013 Behringer et al 2014) In addition 101
GNC and GNL overexpression promotes hypocotyl elongation in light-grown 102
seedlings alterations in leaf shape and an increased angle between the 103
primary and lateral inflorescences (Behringer et al 2014) Importantly 104
deletion or mutation of the LLM-domain of GNC or GNL compromises some 105
but not all phenotypes when these deletion or mutant variants are 106
overexpressed in Arabidopsis and the effects of the overexpression 107
transgenes are compared to the effects of the overexpression of the wild type 108
proteins (Behringer et al 2014) The fact that the overexpression of every 109
LLM-domain B-GATA factor tested to date results in highly comparable 110
phenotypes suggested that LLM-domain B-GATAs have conserved 111
biochemical functions (Behringer et al 2014) 112
A range of studies have already addressed the upstream signaling pathways 113
controlling GNC and GNL expression GNC was initially identified as a nitrate-114
inducible gene with an apparent role in the control of greening (Bi et al 2005) 115
GNL was originally identified based on its strong CK- (cytokinin-)regulated 116
gene expression and therefore designated CYTOKININ-RESPONSIVE GATA 117
FACTOR1 (CGA1) (Naito et al 2007) The expression of GNC and GNL is 118
also controlled by the DELLA regulators of the GA (gibberellin) signaling 119
pathway as well as by the light-labile PIFs (PHYTOCHROME INTERACTING 120
FACTORS) of the phytochrome signaling pathway which are themselves 121
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
5
negatively regulated by DELLAs (Richter et al 2010 Leivar and Monte 122
2014) Furthermore the auxin signaling regulators ARF2 (AUXIN RESPONSE 123
FACTOR2) and ARF7 the flowering regulator SOC1 (SUPPRESSOR-OF-124
OVEREXPRESSION-OF-CONSTANS1) as well as the floral development 125
regulators AP3 (APETALA3) and (PI) PISTILLATA have been implicated in 126
GNC and GNL transcription control (Mara and Irish 2008 Richter et al 2010 127
Richter et al 2013 Richter et al 2013) Thus multiple signaling pathways 128
converge on GNC and GNL expression and the two genes control a broad 129
range of developmental responses in plants 130
To date nothing is known about the regulation and biological function of the 131
four Arabidopsis LLM-domain B-GATAs GATA15 GATA16 GATA17 and 132
GATA17L Here we examine the entire LLM-domain B-GATA gene family 133
through the analysis of single gene insertion mutants and a range of mutant 134
combinations These analyses reveal mainly overlapping roles for these B-135
GATAs in the control of previously known B-GATA-regulated processes such 136
as greening hypocotyl elongation flowering time and senescence but they 137
also reveal previously unknown functions of LLM-domain B-GATAs in 138
phyllotactic patterning floral organ initiation and accessory meristem 139
formation Several of these responses may be mediated by CK signaling and 140
CK treatments induce the expression of all six GATA genes CK-induced gene 141
expression is partially compromised in LLM-domain B-GATA mutants 142
suggesting that B-GATA genes play a role in CK responses Furthermore we 143
provide evidence for a cross-regulation of the different LLM-domain B-GATA 144
genes at the transcriptional level 145
146
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
6
RESULTS 147
Evolutionary conservation of the LLM-domain B-GATAs within the 148
Brassicaceae The Arabidopsis thaliana genome encodes six LLM-domain 149
GATA transcription factors namely GNC (AtGATA21) GNL (AtGATA22) as 150
well as GATA15 (AtGATA15) GATA16 (AtGATA16) GATA17 (AtGATA17) 151
and GATA17L (AtGATA17-LIKE) (Behringer et al 2014 Behringer and 152
Schwechheimer 2015) Phylogenetic analyses showed that these proteins 153
can be grouped into three pairs of highly homologous proteins comprised of 154
the with regard to the length of their N-termini long B-GATAs GNC and GNL 155
and the short B-GATAs GATA15 and GATA16 as well as GATA17 and 156
GATA17L respectively (Fig 1A and B Fig S1) (Behringer et al 2014 157
Behringer and Schwechheimer 2015) It had previously become apparent 158
that the respective subfamilies of long and short B-GATAs were conserved in 159
different plant species but that gene duplications in individual subfamilies may 160
have led to a differential expansion of these subfamilies in different species 161
(Behringer et al 2014) To gain an insight into the conservation of these 162
GATAs within the family of Brassicaceae we searched for LLM-domain B-163
GATAs in the fully sequenced genomes of Arabidopsis lyrata Capsella 164
rubella Eutrema salsugineum and Brassica rapa We retrieved orthologs for 165
each of the six Arabidopsis B-GATAs within each Brassicaceae species (Fig 166
S1 and Fig S2) Whereas the Arabidopsis lyrata Capsella rubella and 167
Eutrema salsugineum genomes contained exactly one apparent ortholog for 168
each of the six Arabidopsis thaliana genes the Brassica rapa genome known 169
to have undergone hexaploidization followed by genome fractionation 170
retained at least two orthologs each for GNC GNL GATA17 and GATA17L 171
and one ortholog of GATA15 and GATA16 (Fig S1 and Fig S2) (Wang et al 172
2011 Tang et al 2012) In summary we concluded that the six LLM-domain 173
B-GATAs are conserved in the different Brassicaceae examined here and that 174
four members of the gene family were present in multiple copies in Brassica 175
rapa 176
177
Characterization of insertion mutants of the six LLM-domain B-GATA 178
genes All previously published genetic analyses of the LLM-domain B-179
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
7
GATAs genes from Arabidopsis thaliana focused on GNC and GNL 180
(Behringer and Schwechheimer 2015) As yet no mutants have been 181
described for the remaining four LLM-domain B-GATAs and their biological 182
function remains elusive We therefore identified homozygous lines of T-DNA 183
insertion mutants for each of the six genes (Fig 1C Fig S3) and 184
subsequently analyzed these lines with regard to the effects of the respective 185
mutations on gene expression using quantitative real time PCR (qRT-PCR) 186
and semi-quantitative PCR (sqRT-PCR) (Fig S3B) The analysis of the gnc 187
allele (Salk_001778) which had already been used in several publications 188
suggested that neither the full-length transcript nor regions downstream from 189
the insertion site were transcribed (Fig 1C and Fig S3B) (Richter et al 2010) 190
(Bi et al 2005) Similarly analyses of the gnl insertion mutant (Salk_003995) 191
using primers spanning the entire transcript or flanking the T-DNA harbored in 192
the intron suggested that the GNL full-length transcript was absent (Fig 1C 193
and Fig S3B) (Bi et al 2005 Richter et al 2010) Although one of the 194
selected GATA15 insertion alleles (gata15-1 SAIL_618_B11) was annotated 195
as an in-gene insertion (wwwsignalsalkedu) we found no evidence for a 196
change of GATA15 gene expression in the homozygous insertion line (Fig 1C 197
and Fig S3B) Sequencing of the insertion site then revealed that the 198
insertion was downstream from the genersquos 3-UTR (untranslated region) 199
indicating that the GATA15 gene was intact in SAIL_618_B11 (Fig 1C and 200
Fig S3B) The analysis of a second allele (gata15-2 WiscDsLox471A10) with 201
an exon-insertion revealed a strong down-regulation of GATA15 expression in 202
both RT-PCR analyses (Fig 1C and Fig S3B) In a GATA16 allele with a T-203
DNA insertion in the genersquos 3-UTR (gata16-1 Salk_021471) transcript 204
abundance was partially reduced (Fig 1C and Fig S3B) The abundance of 205
GATA17 was also partially reduced in Salk_101994 (gata17-1) which 206
harbored a T-DNA insertion in the genes 5-UTR and strongly reduced in 207
SALK_049041 (gata17-2) which carried an insertion in the LLM-domain-208
encoding gene region (Fig 1C and Fig S3B) Finally we detected no 209
transcript in our analyses of a GATA17L exon insertion allele (gata17l-1 210
Salk_026798) when using primer pairs spanning the insertion or located 211
downstream from the insertion site (Fig 1C and Fig S3B) When analyzing 212
this allele with a primer combination upstream of the insertion we were able 213
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
8
to detect residual transcript in gata17l-1 but judged that this may not give rise 214
to a fully functional protein since the insertion was upstream of the LLM-215
domain (Fig 1C and Fig S3B) We concluded that the expression of the 216
respective GATA genes was absent or at least strongly reduced in alleles for 217
GNC (gnc) GNL (gnl) GATA15 (gata15-2) GATA17 (gata17-2) and 218
GATA17L (gata17l-1) alleles The gata16-1 and gata17-1 alleles were only 219
partially impaired in the expression of the respective genes whereas the 220
gata15-1 allele was not affected in GATA15 expression The gata15-2 and 221
gata17-2 alleles became available to us only later in this study and for 222
strategic reasons could not be included in the generation of the complex 223
mutant combinations described below Any of the phenotypes described in the 224
following sections for the more complex mutant combinations were not 225
apparent in the gata15-2 and gata17-2 single mutants We therefore exclude 226
the possibility that these genes are by themselves responsible for any of the 227
phenotypes described here for the higher order mutants 228
229
LLM-domain B-GATAs redundantly control greening We did not observe 230
any obvious defects in the gata single mutants apart from the already well-231
documented defects in greening of the gnc mutants (Bi et al 2005 Richter et 232
al 2010 Chiang et al 2012) To generate higher order mutants in the 233
closely related B-GATA genes we combined the alleles gnc gnl gata15-1 234
gata16-1 gata17-1 and gata17l-1 For simplicity we will subsequently omit 235
the respective allele numbers Since the presence of the misannotated 236
gata15-1 mutation does not provide information about the loss of GATA15 we 237
refer to this allele as GATA15 238
Through genetic crosses we obtained gata17 gata17l double mutants 239
GATA15 gata16 gata17 gata17l gnc gnl GATA15 gata16 and gnc gnl gata17 240
gata17l triple and quadruple mutants as well as a gnc gnl GATA15 gata16 241
gata17 gata17l quintuple mutant In the following we will refer to the gata 242
quintuple mutant as the quintuple mutant and to the GATA15 gata16 gata17 243
gata17l triple mutant that does not include the gnc and gnl alleles as the 244
(complementary) triple mutant In all other cases we will specify the allele 245
combinations 246
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
9
To examine a possible redundant function among the B-GATAs in the control 247
of greening we analyzed greening and chlorophyll content in 14 day-old light-248
grown gata mutant plants (Fig 1D and E) Although there was no indication 249
for a defect in greening in gata17 gata17l or in the triple mutant we found that 250
the greening defect of gnc gnl was further enhanced in the gnc gnl gata17 251
gata17l quadruple mutant and even more in the quintuple mutant (Fig 1D and 252
E) At the same time greening defects were not obvious in any of the gata 253
single mutants (Fig 1E) In the case of the gata15 and gata17 alleles this 254
was not due to the fact that weak alleles had been chosen for the complex 255
mutants since also the severe alleles gata15-2 and gata17-2 showed no 256
reduction in chlorophyll levels when compared to the wild type (Fig 1E) 257
The further reduction in chlorophyll accumulation in the quintuple mutant was 258
also apparent when we analyzed chlorophyll abundance by fluorescence 259
microscopy where we found reduced chloroplast fluorescence when 260
comparing the quintuple mutant to gnc gnl (Fig 1F) Furthermore we noted 261
that the greening defect gradually disappeared in gnc gnl mutants after the 262
seedling stage but remained apparent in the quintuple mutants also in adult 263
plants (Fig S4) Taken together this suggested that all mutated GATA genes 264
participate in the control of greening and chlorophyll accumulation and that 265
the defects of mutants defective in a single gene may be suppressed by the 266
presence of the other family members 267
268
Gene expression regulation of the LLM-domain B-GATAs GNC and GNL 269
had been in the focus of several studies based on the strong regulation of 270
their gene expression by light and CK (Bi et al 2005 Naito et al 2007 271
Richter et al 2010 Richter et al 2013) We were therefore interested in 272
evaluating to what extent light and CK regulate the expression of the other 273
GATA genes In the case of light regulation we found that far-red red as well 274
as blue light strongly induced the expression of GNL and to some extent also 275
the expression of GNC (Fig 2) In blue light we noted a subtle but significant 276
induction of GATA15 and GATA16 (Fig 2C) Importantly this regulation was 277
not observed in the red and far-red light receptor mutant phyA phyB or in the 278
blue light receptor mutant cry1 cry2 (Fig 2) Furthermore light-induced gene 279
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
10
expression changes in red light were impaired in the quadruple mutant for the 280
PHYTOCHROME INTERACTING FACTOR (PIF) genes PIF1 PIF3 PIF4 281
and PIF5 (pifq) indicating that the light-dependent regulation in red light was 282
mediated by the phytochrome and PIF signaling pathways (Fig S5) (Leivar et 283
al 2008) 284
The CK experiments indicated that CK induces the expression of all six GATA 285
genes (Fig 3) With an about three-fold increase in transcript abundance this 286
induction was strongest for GNL but could be observed for each of the other 287
GATA genes after a one to four hour CK treatment (Fig 3) Thus CK 288
promotes the expression of all six LLM-domain B-GATAs 289
290
Mutants of LLM-domain B-GATAs are impaired in hypocotyl elongation 291
Due to the strong regulation particularly of GNL but also of GNC in far-red red 292
and blue light conditions we examined the contribution of the GATA genes to 293
hypocotyl elongation by analyzing mutant seedlings in the different light 294
conditions in two different light intensities (Fig 4) Here we found that 295
quintuple mutant seedlings had a shorter hypocotyl than wild type seedlings in 296
white light as well as in red far-red and blue light (Fig 4) Notably these 297
phenotypes were not apparent or at least not significant in the gnc gnl 298
double mutant or the triple mutant suggesting that the respective other GATA 299
genes may contribute to the hypocotyl elongation phenotype in these 300
backgrounds (Fig 4) At the same time hypocotyl length of dark-grown 301
mutant seedlings was indistinguishable in all genotypes from that observed in 302
the wild type (Fig 4E and G) The short hypocotyl phenotype of the GATA 303
gene mutants was also interesting because we previously observed that the 304
LLM-domain B-GATA overexpressors had a longer hypocotyl than the wild 305
type when grown in white light (Behringer et al 2014) When analyzed in the 306
different light conditions we found the long hypocotyl phenotype of the GNL 307
overexpression line (GNLox) was also present in far-red and blue light but 308
interestingly not in red light where hypocotyls were even shorter than in the 309
wild type or the mutants when grown in weak red light conditions (Fig 4) 310
311
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
11
GATA gene mutants have differential gene expression defects The 312
strong induction of GNL gene expression by CK was already repeatedly 313
described and is the reason for the genersquos original designation CGA1 314
(CYTOKININ-INDUCED GATA1) (Naito et al 2007 Kollmer et al 2011) In 315
order to examine the role of the GATA genes in CK-responsive gene 316
expression we performed microarray experiments with 14 day-old wild type 317
plants gnc gnl and triple mutants as well as quintuple mutants treated for 60 318
min with the CK 6-BA (6-benzylaminopurine) or a corresponding mock solvent 319
control (Fig 5 and Fig 6 Supplemental Tables S1 and S2) We then 320
analyzed the resulting data with regard to differences in basal gene 321
expression (Fig 5) and with regard to gene expression differences after the 322
CK treatment in comparison to the mock control (Fig 6) For technical 323
reasons the experiment was conducted in two rounds the first experiment 324
included the Col-0 (1) wild type control the gnc gnl double mutant and the 325
quintuple mutant the second experiment included an independent Col-0 (2) 326
wild type control and the triple mutant When we examined the expression of 327
the CK-induced ARRs (A-TYPE RESPONSE REGULATORS) of the CK 328
signaling pathway we could confirm that the respective CK induction 329
experiments were comparably efficient between the two experiments and in 330
all genotypes tested (Fig S6A) We also examined the microarray dataset 331
with regard to the expression of a previously defined so-called ldquogolden listrdquo of 332
CK-regulated genes (Bhargava et al 2013) Over 70 of the 331 entities 333
derived from this list were significantly CK-regulated in our experiments 334
(Supplemental Table S3) When applying a two-fold expression change as 335
additional filter criterion 71 of these 331 entities were identified as up- (Col-0 336
1 and Col-0 2) and 39 (Col-0 1) and 25 entities (Col-0 2) as down-regulated 337
after CK treatment (Fig S6B and Supplemental Table S3) We thus 338
concluded that the CK treatments in both experiments were similarly efficient 339
and that the data sets could be used for comparative analyses 340
The comparison of the basal gene expression profiles of the wild type 341
samples and the mutants indicated that the (untreated) quintuple mutant 342
seedlings had by far the largest number of differentially regulated genes with 343
4722 down-regulated and 976 up-regulated entities when compared to the 344
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
12
Col-0 (1) wild type (Fig 5 and Supplemental Table S1) Furthermore the gnc 345
gnl double mutant contained 2458 down- and 294 up-regulated entities 346
respectively (Fig 5 and Supplemental Table S1) In comparison the number 347
of differentially expressed genes was comparatively minor in the triple mutant 348
which had only 323 down- and 278 up-regulated entities in this particular 349
experiment and selected physiological growth conditions (Fig 5) 350
The comparatively strong gene expression differences in the gnc gnl double 351
and the quintuple mutants and the fact that both mutants shared the gnc gnl 352
loss-of-function at the genetic level was reflected by the large overlap 353
between the differentially expressed gene sets of the two genotypes 354
Specifically 1922 of the 2458 down-regulated entities from gnc gnl were also 355
down-regulated in the quintuple mutant (Fig 5B) and 98 of the 294 entities 356
up-regulated in gnc gnl were also up-regulated in the quintuple mutant (Fig 357
5B) Conversely the overlap in gene expression defects between the triple 358
mutant and the quintuple mutant was higher than that between the triple 359
mutant and gnc gnl (Fig 5B) We therefore concluded that the gene 360
expression differences between the different GATA gene mutants was 361
strongest in the quintuple mutant and that the strong increase in the number 362
of differentially regulated genes exceeded by far the differences seen in the 363
gnc gnl double mutant and the triple mutant This supported our conclusion 364
from the phenotypic analyses that the GATA genes act in a functionally 365
redundant manner 366
Since we had observed increased gene expression of all six GATA genes 367
after CK-treatment we expected that CK-induced gene expression may be 368
compromised in the gata mutants (Fig 6) Indeed of the 996 entities that 369
were down-regulated after CK-treatment in the wild type (Col-0 1) only 173 370
(18 ) and 89 (9 ) remained CK-regulated in the quintuple and gnc gnl 371
mutant respectively (Fig 6A and Supplemental Table S2) At the same time 372
of the 188 entities that were down-regulated after CK treatment in the wild 373
type sample of the second experiment (Col-0 2) 83 (44 ) remained CK-374
regulated in the gata triple mutant (Fig 6B) The differential effect on gene 375
expression between the different mutants was less pronounced when 376
examining the CK-induced genes Here 49 (quintuple mutant) 59 (gnc 377
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
13
gnl) and 63 (triple mutant) of the CK-induced entities from the respective 378
wild types remained CK-regulated in the mutants (Fig 6A and B) In summary 379
we concluded that CK-regulated gene expression is impaired in the GATA 380
gene mutants 381
382
GATA gene mutants are defective in CK-regulated developmental 383
processes Based on the strong defects of gata mutants in CK-regulated 384
gene expression we searched for phenotypes that could be explained by 385
defects in CK response CK signaling was described as a regulator of 386
phyllotactic patterning in the shoot apical meristem of Arabidopsis (Besnard et 387
al 2014) In line with a role of the LLM-domain B-GATA genes in this process 388
we observed frequent aberrations from normal phyllotactic patterning in flower 389
positioning in triple and quintuple mutant inflorescences (Fig 7) Quantitative 390
analyses revealed aberrations from the canonical angle of ~1375deg in about 391
~30 of the petioles in triple and quintuple inflorescences (Fig 7D) At the 392
same time this patterning defect was not apparent in the gnc gnl double 393
mutant Since we also did not observe a quantitative increase in the severity 394
of this phenotype between the triple and the quintuple mutant we concluded 395
that GATA16 GATA17 and GATA17L may be responsible for this phenotype 396
(Fig 7D) 397
Leaf senescence is another CK-regulated process (Hwang et al 2012) In the 398
wild type CK levels are reduced during senescence and positive or negative 399
interference with the decrease in CK levels can promote and delay 400
senescence respectively (Hwang et al 2012) Senescence can be induced 401
by shifting light-grown plants to the dark and we had previously already shown 402
that leaf senescence is delayed in overexpression lines of LLM-domain B-403
GATAs (Behringer et al 2014) To examine possible senescence defects in 404
the gata mutants we floated leaves of wild type and mutant plants in the dark 405
on a mock solution or on a solution containing 005 microM or 01 microM 6-BA (Fig 406
8A ndash F) (Weaver and Amasino 2001) In line with the expectation that the 407
presence of CK in the medium would suppress the senescence process we 408
observed decreased senescence in CK-treated wild type leaves which we 409
quantitatively assessed by measuring chlorophyll levels in the mock- and CK-410
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
14
treated leaves (Fig 8F) Here we found that the effects of the senescence 411
treatment were less efficiently suppressed by CK treatments in the gnc gnl 412
double and in the quintuple mutant than in the wild type or the triple mutant 413
(Fig 8A - F) Similarly we observed a stronger effect on the decreases in 414
chlorophyll and total protein levels in the gnc gnl as well as the quintuple 415
mutant when we transferred light-grown plants for four days to the dark (Fig 416
8G ndash I) As previously reported GNL overexpressing plants (GNLox) had 417
senescence phenotypes that were antagonistic to the ones observed with the 418
mutants (Fig 8) (Behringer et al 2014) In the case of the leaf floating assay 419
CK-treatment had a stronger effect on greening and chlorophyll abundance in 420
GNLox than in the wild type (Fig 8A ndash F) When we transferred plants from 421
the light to the dark GNLox seedlings remained visibly green and did not 422
display the strong decrease in total protein abundance that we observed in 423
the wild type or the GATA gene mutants (Fig 8G ndash I) We thus concluded that 424
GATA gene mutants and overexpressors have antagonistic effects with 425
regard to the regulation of senescence and that GNC and GNL contribute 426
particularly strongly to this phenotype 427
CK induces greening in light-grown seedlings (Kobayashi et al 2012) To 428
examine to what extent CK can induce chlorophyll accumulation in the gata 429
mutants we grew plants for two weeks in constant light on medium containing 430
CK (5 nM BA) or a corresponding CK-free medium As expected chlorophyll 431
content was increased in wild type plants but this increase was neither 432
significant in the gnc gnl nor the triple or the quintuple mutants (Fig S7) We 433
thus concluded that the GATA factors are required for CK-induced greening in 434
Arabidopsis seedlings 435
It was previously reported that hypocotyl elongation could be repressed by 436
incubating dark-grown seedlings in the presence of CK (Su and Howell 1995) 437
Since the GATA gene mutants had displayed a hypocotyl elongation defect in 438
light-grown seedlings (Fig 4) we also explored the link between hypocotyl 439
elongation and CK However when we tested the effects of CK on hypocotyl 440
elongation in dark-grown seedlings we did not detect any significant 441
differences in the response between the different genotypes tested (Fig S8) 442
It is thus unlikely that the GATA genes participate in this elongation response 443
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
15
Branching is another phenotype that has been attributed to defects in CK 444
biosynthesis and response (El-Showk et al 2013) When we analyzed the 445
general habitus of the quintuple mutants we noted that they had an increased 446
number of secondary branches that was combined with a reduced plant 447
height (Fig 9A - C) Occasionally we also observed the outgrowth of 448
inflorescences in the axils of lateral inflorescences in the mutants whereas we 449
had not noted this phenotype in the wild type (Fig 9D and E) A quantitative 450
analysis of these phenotypes over all gata mutant phenotypes revealed that 451
the number of first and second order lateral branches increased with 452
increasing mutant complexity (Fig 9B) Here we observed strong effects 453
already in the gata17 gata17l mutant as well as the gnc gnl double mutant but 454
strongest defects in the triple and the quintuple mutant thus all five GATA 455
genes may contribute to this phenotype (Fig 9B) At the same time plant 456
height was reduced to a significant but still minor level in these mutants 457
suggesting that decreases in plant height which may have indirect 458
consequences on the general plant habitus and branching are not the 459
primary cause for altered branching patterns in the gata mutants (Fig 9C) It 460
is further important to note that the higher order GATA gene mutants initiated 461
fewer vegetative buds but had a higher number of growing buds as reflected 462
by the increased branch numbers (Fig 9F) Thus axillary bud formation is 463
promoted and bud outgrowth is repressed by the GATAs in the wild type 464
Another phenotype that became apparent during our analysis of the gata 465
mutant plant habitus was an increase in the angle formed between the 466
primary inflorescence and the lateral inflorescences (Fig 9G and H Fig S9) 467
gata mutants had a decreased angle formed between the primary 468
inflorescence and lateral inflorescences and this phenotype was strongest in 469
the quintuple mutant and intermediate in the gnc gnl and triple mutants (Fig 470
9G and H Fig S9) Interestingly we had previously observed increased 471
branch angles in GATA overexpression lines such as GNLox (Fig S9) 472
(Behringer et al 2014) We thus concluded that GATA gene mutants and 473
overexpressors antagonistically regulate branch angles in Arabidopsis 474
475
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
5
negatively regulated by DELLAs (Richter et al 2010 Leivar and Monte 122
2014) Furthermore the auxin signaling regulators ARF2 (AUXIN RESPONSE 123
FACTOR2) and ARF7 the flowering regulator SOC1 (SUPPRESSOR-OF-124
OVEREXPRESSION-OF-CONSTANS1) as well as the floral development 125
regulators AP3 (APETALA3) and (PI) PISTILLATA have been implicated in 126
GNC and GNL transcription control (Mara and Irish 2008 Richter et al 2010 127
Richter et al 2013 Richter et al 2013) Thus multiple signaling pathways 128
converge on GNC and GNL expression and the two genes control a broad 129
range of developmental responses in plants 130
To date nothing is known about the regulation and biological function of the 131
four Arabidopsis LLM-domain B-GATAs GATA15 GATA16 GATA17 and 132
GATA17L Here we examine the entire LLM-domain B-GATA gene family 133
through the analysis of single gene insertion mutants and a range of mutant 134
combinations These analyses reveal mainly overlapping roles for these B-135
GATAs in the control of previously known B-GATA-regulated processes such 136
as greening hypocotyl elongation flowering time and senescence but they 137
also reveal previously unknown functions of LLM-domain B-GATAs in 138
phyllotactic patterning floral organ initiation and accessory meristem 139
formation Several of these responses may be mediated by CK signaling and 140
CK treatments induce the expression of all six GATA genes CK-induced gene 141
expression is partially compromised in LLM-domain B-GATA mutants 142
suggesting that B-GATA genes play a role in CK responses Furthermore we 143
provide evidence for a cross-regulation of the different LLM-domain B-GATA 144
genes at the transcriptional level 145
146
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
6
RESULTS 147
Evolutionary conservation of the LLM-domain B-GATAs within the 148
Brassicaceae The Arabidopsis thaliana genome encodes six LLM-domain 149
GATA transcription factors namely GNC (AtGATA21) GNL (AtGATA22) as 150
well as GATA15 (AtGATA15) GATA16 (AtGATA16) GATA17 (AtGATA17) 151
and GATA17L (AtGATA17-LIKE) (Behringer et al 2014 Behringer and 152
Schwechheimer 2015) Phylogenetic analyses showed that these proteins 153
can be grouped into three pairs of highly homologous proteins comprised of 154
the with regard to the length of their N-termini long B-GATAs GNC and GNL 155
and the short B-GATAs GATA15 and GATA16 as well as GATA17 and 156
GATA17L respectively (Fig 1A and B Fig S1) (Behringer et al 2014 157
Behringer and Schwechheimer 2015) It had previously become apparent 158
that the respective subfamilies of long and short B-GATAs were conserved in 159
different plant species but that gene duplications in individual subfamilies may 160
have led to a differential expansion of these subfamilies in different species 161
(Behringer et al 2014) To gain an insight into the conservation of these 162
GATAs within the family of Brassicaceae we searched for LLM-domain B-163
GATAs in the fully sequenced genomes of Arabidopsis lyrata Capsella 164
rubella Eutrema salsugineum and Brassica rapa We retrieved orthologs for 165
each of the six Arabidopsis B-GATAs within each Brassicaceae species (Fig 166
S1 and Fig S2) Whereas the Arabidopsis lyrata Capsella rubella and 167
Eutrema salsugineum genomes contained exactly one apparent ortholog for 168
each of the six Arabidopsis thaliana genes the Brassica rapa genome known 169
to have undergone hexaploidization followed by genome fractionation 170
retained at least two orthologs each for GNC GNL GATA17 and GATA17L 171
and one ortholog of GATA15 and GATA16 (Fig S1 and Fig S2) (Wang et al 172
2011 Tang et al 2012) In summary we concluded that the six LLM-domain 173
B-GATAs are conserved in the different Brassicaceae examined here and that 174
four members of the gene family were present in multiple copies in Brassica 175
rapa 176
177
Characterization of insertion mutants of the six LLM-domain B-GATA 178
genes All previously published genetic analyses of the LLM-domain B-179
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
7
GATAs genes from Arabidopsis thaliana focused on GNC and GNL 180
(Behringer and Schwechheimer 2015) As yet no mutants have been 181
described for the remaining four LLM-domain B-GATAs and their biological 182
function remains elusive We therefore identified homozygous lines of T-DNA 183
insertion mutants for each of the six genes (Fig 1C Fig S3) and 184
subsequently analyzed these lines with regard to the effects of the respective 185
mutations on gene expression using quantitative real time PCR (qRT-PCR) 186
and semi-quantitative PCR (sqRT-PCR) (Fig S3B) The analysis of the gnc 187
allele (Salk_001778) which had already been used in several publications 188
suggested that neither the full-length transcript nor regions downstream from 189
the insertion site were transcribed (Fig 1C and Fig S3B) (Richter et al 2010) 190
(Bi et al 2005) Similarly analyses of the gnl insertion mutant (Salk_003995) 191
using primers spanning the entire transcript or flanking the T-DNA harbored in 192
the intron suggested that the GNL full-length transcript was absent (Fig 1C 193
and Fig S3B) (Bi et al 2005 Richter et al 2010) Although one of the 194
selected GATA15 insertion alleles (gata15-1 SAIL_618_B11) was annotated 195
as an in-gene insertion (wwwsignalsalkedu) we found no evidence for a 196
change of GATA15 gene expression in the homozygous insertion line (Fig 1C 197
and Fig S3B) Sequencing of the insertion site then revealed that the 198
insertion was downstream from the genersquos 3-UTR (untranslated region) 199
indicating that the GATA15 gene was intact in SAIL_618_B11 (Fig 1C and 200
Fig S3B) The analysis of a second allele (gata15-2 WiscDsLox471A10) with 201
an exon-insertion revealed a strong down-regulation of GATA15 expression in 202
both RT-PCR analyses (Fig 1C and Fig S3B) In a GATA16 allele with a T-203
DNA insertion in the genersquos 3-UTR (gata16-1 Salk_021471) transcript 204
abundance was partially reduced (Fig 1C and Fig S3B) The abundance of 205
GATA17 was also partially reduced in Salk_101994 (gata17-1) which 206
harbored a T-DNA insertion in the genes 5-UTR and strongly reduced in 207
SALK_049041 (gata17-2) which carried an insertion in the LLM-domain-208
encoding gene region (Fig 1C and Fig S3B) Finally we detected no 209
transcript in our analyses of a GATA17L exon insertion allele (gata17l-1 210
Salk_026798) when using primer pairs spanning the insertion or located 211
downstream from the insertion site (Fig 1C and Fig S3B) When analyzing 212
this allele with a primer combination upstream of the insertion we were able 213
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
8
to detect residual transcript in gata17l-1 but judged that this may not give rise 214
to a fully functional protein since the insertion was upstream of the LLM-215
domain (Fig 1C and Fig S3B) We concluded that the expression of the 216
respective GATA genes was absent or at least strongly reduced in alleles for 217
GNC (gnc) GNL (gnl) GATA15 (gata15-2) GATA17 (gata17-2) and 218
GATA17L (gata17l-1) alleles The gata16-1 and gata17-1 alleles were only 219
partially impaired in the expression of the respective genes whereas the 220
gata15-1 allele was not affected in GATA15 expression The gata15-2 and 221
gata17-2 alleles became available to us only later in this study and for 222
strategic reasons could not be included in the generation of the complex 223
mutant combinations described below Any of the phenotypes described in the 224
following sections for the more complex mutant combinations were not 225
apparent in the gata15-2 and gata17-2 single mutants We therefore exclude 226
the possibility that these genes are by themselves responsible for any of the 227
phenotypes described here for the higher order mutants 228
229
LLM-domain B-GATAs redundantly control greening We did not observe 230
any obvious defects in the gata single mutants apart from the already well-231
documented defects in greening of the gnc mutants (Bi et al 2005 Richter et 232
al 2010 Chiang et al 2012) To generate higher order mutants in the 233
closely related B-GATA genes we combined the alleles gnc gnl gata15-1 234
gata16-1 gata17-1 and gata17l-1 For simplicity we will subsequently omit 235
the respective allele numbers Since the presence of the misannotated 236
gata15-1 mutation does not provide information about the loss of GATA15 we 237
refer to this allele as GATA15 238
Through genetic crosses we obtained gata17 gata17l double mutants 239
GATA15 gata16 gata17 gata17l gnc gnl GATA15 gata16 and gnc gnl gata17 240
gata17l triple and quadruple mutants as well as a gnc gnl GATA15 gata16 241
gata17 gata17l quintuple mutant In the following we will refer to the gata 242
quintuple mutant as the quintuple mutant and to the GATA15 gata16 gata17 243
gata17l triple mutant that does not include the gnc and gnl alleles as the 244
(complementary) triple mutant In all other cases we will specify the allele 245
combinations 246
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
9
To examine a possible redundant function among the B-GATAs in the control 247
of greening we analyzed greening and chlorophyll content in 14 day-old light-248
grown gata mutant plants (Fig 1D and E) Although there was no indication 249
for a defect in greening in gata17 gata17l or in the triple mutant we found that 250
the greening defect of gnc gnl was further enhanced in the gnc gnl gata17 251
gata17l quadruple mutant and even more in the quintuple mutant (Fig 1D and 252
E) At the same time greening defects were not obvious in any of the gata 253
single mutants (Fig 1E) In the case of the gata15 and gata17 alleles this 254
was not due to the fact that weak alleles had been chosen for the complex 255
mutants since also the severe alleles gata15-2 and gata17-2 showed no 256
reduction in chlorophyll levels when compared to the wild type (Fig 1E) 257
The further reduction in chlorophyll accumulation in the quintuple mutant was 258
also apparent when we analyzed chlorophyll abundance by fluorescence 259
microscopy where we found reduced chloroplast fluorescence when 260
comparing the quintuple mutant to gnc gnl (Fig 1F) Furthermore we noted 261
that the greening defect gradually disappeared in gnc gnl mutants after the 262
seedling stage but remained apparent in the quintuple mutants also in adult 263
plants (Fig S4) Taken together this suggested that all mutated GATA genes 264
participate in the control of greening and chlorophyll accumulation and that 265
the defects of mutants defective in a single gene may be suppressed by the 266
presence of the other family members 267
268
Gene expression regulation of the LLM-domain B-GATAs GNC and GNL 269
had been in the focus of several studies based on the strong regulation of 270
their gene expression by light and CK (Bi et al 2005 Naito et al 2007 271
Richter et al 2010 Richter et al 2013) We were therefore interested in 272
evaluating to what extent light and CK regulate the expression of the other 273
GATA genes In the case of light regulation we found that far-red red as well 274
as blue light strongly induced the expression of GNL and to some extent also 275
the expression of GNC (Fig 2) In blue light we noted a subtle but significant 276
induction of GATA15 and GATA16 (Fig 2C) Importantly this regulation was 277
not observed in the red and far-red light receptor mutant phyA phyB or in the 278
blue light receptor mutant cry1 cry2 (Fig 2) Furthermore light-induced gene 279
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
10
expression changes in red light were impaired in the quadruple mutant for the 280
PHYTOCHROME INTERACTING FACTOR (PIF) genes PIF1 PIF3 PIF4 281
and PIF5 (pifq) indicating that the light-dependent regulation in red light was 282
mediated by the phytochrome and PIF signaling pathways (Fig S5) (Leivar et 283
al 2008) 284
The CK experiments indicated that CK induces the expression of all six GATA 285
genes (Fig 3) With an about three-fold increase in transcript abundance this 286
induction was strongest for GNL but could be observed for each of the other 287
GATA genes after a one to four hour CK treatment (Fig 3) Thus CK 288
promotes the expression of all six LLM-domain B-GATAs 289
290
Mutants of LLM-domain B-GATAs are impaired in hypocotyl elongation 291
Due to the strong regulation particularly of GNL but also of GNC in far-red red 292
and blue light conditions we examined the contribution of the GATA genes to 293
hypocotyl elongation by analyzing mutant seedlings in the different light 294
conditions in two different light intensities (Fig 4) Here we found that 295
quintuple mutant seedlings had a shorter hypocotyl than wild type seedlings in 296
white light as well as in red far-red and blue light (Fig 4) Notably these 297
phenotypes were not apparent or at least not significant in the gnc gnl 298
double mutant or the triple mutant suggesting that the respective other GATA 299
genes may contribute to the hypocotyl elongation phenotype in these 300
backgrounds (Fig 4) At the same time hypocotyl length of dark-grown 301
mutant seedlings was indistinguishable in all genotypes from that observed in 302
the wild type (Fig 4E and G) The short hypocotyl phenotype of the GATA 303
gene mutants was also interesting because we previously observed that the 304
LLM-domain B-GATA overexpressors had a longer hypocotyl than the wild 305
type when grown in white light (Behringer et al 2014) When analyzed in the 306
different light conditions we found the long hypocotyl phenotype of the GNL 307
overexpression line (GNLox) was also present in far-red and blue light but 308
interestingly not in red light where hypocotyls were even shorter than in the 309
wild type or the mutants when grown in weak red light conditions (Fig 4) 310
311
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
11
GATA gene mutants have differential gene expression defects The 312
strong induction of GNL gene expression by CK was already repeatedly 313
described and is the reason for the genersquos original designation CGA1 314
(CYTOKININ-INDUCED GATA1) (Naito et al 2007 Kollmer et al 2011) In 315
order to examine the role of the GATA genes in CK-responsive gene 316
expression we performed microarray experiments with 14 day-old wild type 317
plants gnc gnl and triple mutants as well as quintuple mutants treated for 60 318
min with the CK 6-BA (6-benzylaminopurine) or a corresponding mock solvent 319
control (Fig 5 and Fig 6 Supplemental Tables S1 and S2) We then 320
analyzed the resulting data with regard to differences in basal gene 321
expression (Fig 5) and with regard to gene expression differences after the 322
CK treatment in comparison to the mock control (Fig 6) For technical 323
reasons the experiment was conducted in two rounds the first experiment 324
included the Col-0 (1) wild type control the gnc gnl double mutant and the 325
quintuple mutant the second experiment included an independent Col-0 (2) 326
wild type control and the triple mutant When we examined the expression of 327
the CK-induced ARRs (A-TYPE RESPONSE REGULATORS) of the CK 328
signaling pathway we could confirm that the respective CK induction 329
experiments were comparably efficient between the two experiments and in 330
all genotypes tested (Fig S6A) We also examined the microarray dataset 331
with regard to the expression of a previously defined so-called ldquogolden listrdquo of 332
CK-regulated genes (Bhargava et al 2013) Over 70 of the 331 entities 333
derived from this list were significantly CK-regulated in our experiments 334
(Supplemental Table S3) When applying a two-fold expression change as 335
additional filter criterion 71 of these 331 entities were identified as up- (Col-0 336
1 and Col-0 2) and 39 (Col-0 1) and 25 entities (Col-0 2) as down-regulated 337
after CK treatment (Fig S6B and Supplemental Table S3) We thus 338
concluded that the CK treatments in both experiments were similarly efficient 339
and that the data sets could be used for comparative analyses 340
The comparison of the basal gene expression profiles of the wild type 341
samples and the mutants indicated that the (untreated) quintuple mutant 342
seedlings had by far the largest number of differentially regulated genes with 343
4722 down-regulated and 976 up-regulated entities when compared to the 344
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
12
Col-0 (1) wild type (Fig 5 and Supplemental Table S1) Furthermore the gnc 345
gnl double mutant contained 2458 down- and 294 up-regulated entities 346
respectively (Fig 5 and Supplemental Table S1) In comparison the number 347
of differentially expressed genes was comparatively minor in the triple mutant 348
which had only 323 down- and 278 up-regulated entities in this particular 349
experiment and selected physiological growth conditions (Fig 5) 350
The comparatively strong gene expression differences in the gnc gnl double 351
and the quintuple mutants and the fact that both mutants shared the gnc gnl 352
loss-of-function at the genetic level was reflected by the large overlap 353
between the differentially expressed gene sets of the two genotypes 354
Specifically 1922 of the 2458 down-regulated entities from gnc gnl were also 355
down-regulated in the quintuple mutant (Fig 5B) and 98 of the 294 entities 356
up-regulated in gnc gnl were also up-regulated in the quintuple mutant (Fig 357
5B) Conversely the overlap in gene expression defects between the triple 358
mutant and the quintuple mutant was higher than that between the triple 359
mutant and gnc gnl (Fig 5B) We therefore concluded that the gene 360
expression differences between the different GATA gene mutants was 361
strongest in the quintuple mutant and that the strong increase in the number 362
of differentially regulated genes exceeded by far the differences seen in the 363
gnc gnl double mutant and the triple mutant This supported our conclusion 364
from the phenotypic analyses that the GATA genes act in a functionally 365
redundant manner 366
Since we had observed increased gene expression of all six GATA genes 367
after CK-treatment we expected that CK-induced gene expression may be 368
compromised in the gata mutants (Fig 6) Indeed of the 996 entities that 369
were down-regulated after CK-treatment in the wild type (Col-0 1) only 173 370
(18 ) and 89 (9 ) remained CK-regulated in the quintuple and gnc gnl 371
mutant respectively (Fig 6A and Supplemental Table S2) At the same time 372
of the 188 entities that were down-regulated after CK treatment in the wild 373
type sample of the second experiment (Col-0 2) 83 (44 ) remained CK-374
regulated in the gata triple mutant (Fig 6B) The differential effect on gene 375
expression between the different mutants was less pronounced when 376
examining the CK-induced genes Here 49 (quintuple mutant) 59 (gnc 377
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
13
gnl) and 63 (triple mutant) of the CK-induced entities from the respective 378
wild types remained CK-regulated in the mutants (Fig 6A and B) In summary 379
we concluded that CK-regulated gene expression is impaired in the GATA 380
gene mutants 381
382
GATA gene mutants are defective in CK-regulated developmental 383
processes Based on the strong defects of gata mutants in CK-regulated 384
gene expression we searched for phenotypes that could be explained by 385
defects in CK response CK signaling was described as a regulator of 386
phyllotactic patterning in the shoot apical meristem of Arabidopsis (Besnard et 387
al 2014) In line with a role of the LLM-domain B-GATA genes in this process 388
we observed frequent aberrations from normal phyllotactic patterning in flower 389
positioning in triple and quintuple mutant inflorescences (Fig 7) Quantitative 390
analyses revealed aberrations from the canonical angle of ~1375deg in about 391
~30 of the petioles in triple and quintuple inflorescences (Fig 7D) At the 392
same time this patterning defect was not apparent in the gnc gnl double 393
mutant Since we also did not observe a quantitative increase in the severity 394
of this phenotype between the triple and the quintuple mutant we concluded 395
that GATA16 GATA17 and GATA17L may be responsible for this phenotype 396
(Fig 7D) 397
Leaf senescence is another CK-regulated process (Hwang et al 2012) In the 398
wild type CK levels are reduced during senescence and positive or negative 399
interference with the decrease in CK levels can promote and delay 400
senescence respectively (Hwang et al 2012) Senescence can be induced 401
by shifting light-grown plants to the dark and we had previously already shown 402
that leaf senescence is delayed in overexpression lines of LLM-domain B-403
GATAs (Behringer et al 2014) To examine possible senescence defects in 404
the gata mutants we floated leaves of wild type and mutant plants in the dark 405
on a mock solution or on a solution containing 005 microM or 01 microM 6-BA (Fig 406
8A ndash F) (Weaver and Amasino 2001) In line with the expectation that the 407
presence of CK in the medium would suppress the senescence process we 408
observed decreased senescence in CK-treated wild type leaves which we 409
quantitatively assessed by measuring chlorophyll levels in the mock- and CK-410
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
14
treated leaves (Fig 8F) Here we found that the effects of the senescence 411
treatment were less efficiently suppressed by CK treatments in the gnc gnl 412
double and in the quintuple mutant than in the wild type or the triple mutant 413
(Fig 8A - F) Similarly we observed a stronger effect on the decreases in 414
chlorophyll and total protein levels in the gnc gnl as well as the quintuple 415
mutant when we transferred light-grown plants for four days to the dark (Fig 416
8G ndash I) As previously reported GNL overexpressing plants (GNLox) had 417
senescence phenotypes that were antagonistic to the ones observed with the 418
mutants (Fig 8) (Behringer et al 2014) In the case of the leaf floating assay 419
CK-treatment had a stronger effect on greening and chlorophyll abundance in 420
GNLox than in the wild type (Fig 8A ndash F) When we transferred plants from 421
the light to the dark GNLox seedlings remained visibly green and did not 422
display the strong decrease in total protein abundance that we observed in 423
the wild type or the GATA gene mutants (Fig 8G ndash I) We thus concluded that 424
GATA gene mutants and overexpressors have antagonistic effects with 425
regard to the regulation of senescence and that GNC and GNL contribute 426
particularly strongly to this phenotype 427
CK induces greening in light-grown seedlings (Kobayashi et al 2012) To 428
examine to what extent CK can induce chlorophyll accumulation in the gata 429
mutants we grew plants for two weeks in constant light on medium containing 430
CK (5 nM BA) or a corresponding CK-free medium As expected chlorophyll 431
content was increased in wild type plants but this increase was neither 432
significant in the gnc gnl nor the triple or the quintuple mutants (Fig S7) We 433
thus concluded that the GATA factors are required for CK-induced greening in 434
Arabidopsis seedlings 435
It was previously reported that hypocotyl elongation could be repressed by 436
incubating dark-grown seedlings in the presence of CK (Su and Howell 1995) 437
Since the GATA gene mutants had displayed a hypocotyl elongation defect in 438
light-grown seedlings (Fig 4) we also explored the link between hypocotyl 439
elongation and CK However when we tested the effects of CK on hypocotyl 440
elongation in dark-grown seedlings we did not detect any significant 441
differences in the response between the different genotypes tested (Fig S8) 442
It is thus unlikely that the GATA genes participate in this elongation response 443
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
15
Branching is another phenotype that has been attributed to defects in CK 444
biosynthesis and response (El-Showk et al 2013) When we analyzed the 445
general habitus of the quintuple mutants we noted that they had an increased 446
number of secondary branches that was combined with a reduced plant 447
height (Fig 9A - C) Occasionally we also observed the outgrowth of 448
inflorescences in the axils of lateral inflorescences in the mutants whereas we 449
had not noted this phenotype in the wild type (Fig 9D and E) A quantitative 450
analysis of these phenotypes over all gata mutant phenotypes revealed that 451
the number of first and second order lateral branches increased with 452
increasing mutant complexity (Fig 9B) Here we observed strong effects 453
already in the gata17 gata17l mutant as well as the gnc gnl double mutant but 454
strongest defects in the triple and the quintuple mutant thus all five GATA 455
genes may contribute to this phenotype (Fig 9B) At the same time plant 456
height was reduced to a significant but still minor level in these mutants 457
suggesting that decreases in plant height which may have indirect 458
consequences on the general plant habitus and branching are not the 459
primary cause for altered branching patterns in the gata mutants (Fig 9C) It 460
is further important to note that the higher order GATA gene mutants initiated 461
fewer vegetative buds but had a higher number of growing buds as reflected 462
by the increased branch numbers (Fig 9F) Thus axillary bud formation is 463
promoted and bud outgrowth is repressed by the GATAs in the wild type 464
Another phenotype that became apparent during our analysis of the gata 465
mutant plant habitus was an increase in the angle formed between the 466
primary inflorescence and the lateral inflorescences (Fig 9G and H Fig S9) 467
gata mutants had a decreased angle formed between the primary 468
inflorescence and lateral inflorescences and this phenotype was strongest in 469
the quintuple mutant and intermediate in the gnc gnl and triple mutants (Fig 470
9G and H Fig S9) Interestingly we had previously observed increased 471
branch angles in GATA overexpression lines such as GNLox (Fig S9) 472
(Behringer et al 2014) We thus concluded that GATA gene mutants and 473
overexpressors antagonistically regulate branch angles in Arabidopsis 474
475
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
6
RESULTS 147
Evolutionary conservation of the LLM-domain B-GATAs within the 148
Brassicaceae The Arabidopsis thaliana genome encodes six LLM-domain 149
GATA transcription factors namely GNC (AtGATA21) GNL (AtGATA22) as 150
well as GATA15 (AtGATA15) GATA16 (AtGATA16) GATA17 (AtGATA17) 151
and GATA17L (AtGATA17-LIKE) (Behringer et al 2014 Behringer and 152
Schwechheimer 2015) Phylogenetic analyses showed that these proteins 153
can be grouped into three pairs of highly homologous proteins comprised of 154
the with regard to the length of their N-termini long B-GATAs GNC and GNL 155
and the short B-GATAs GATA15 and GATA16 as well as GATA17 and 156
GATA17L respectively (Fig 1A and B Fig S1) (Behringer et al 2014 157
Behringer and Schwechheimer 2015) It had previously become apparent 158
that the respective subfamilies of long and short B-GATAs were conserved in 159
different plant species but that gene duplications in individual subfamilies may 160
have led to a differential expansion of these subfamilies in different species 161
(Behringer et al 2014) To gain an insight into the conservation of these 162
GATAs within the family of Brassicaceae we searched for LLM-domain B-163
GATAs in the fully sequenced genomes of Arabidopsis lyrata Capsella 164
rubella Eutrema salsugineum and Brassica rapa We retrieved orthologs for 165
each of the six Arabidopsis B-GATAs within each Brassicaceae species (Fig 166
S1 and Fig S2) Whereas the Arabidopsis lyrata Capsella rubella and 167
Eutrema salsugineum genomes contained exactly one apparent ortholog for 168
each of the six Arabidopsis thaliana genes the Brassica rapa genome known 169
to have undergone hexaploidization followed by genome fractionation 170
retained at least two orthologs each for GNC GNL GATA17 and GATA17L 171
and one ortholog of GATA15 and GATA16 (Fig S1 and Fig S2) (Wang et al 172
2011 Tang et al 2012) In summary we concluded that the six LLM-domain 173
B-GATAs are conserved in the different Brassicaceae examined here and that 174
four members of the gene family were present in multiple copies in Brassica 175
rapa 176
177
Characterization of insertion mutants of the six LLM-domain B-GATA 178
genes All previously published genetic analyses of the LLM-domain B-179
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
7
GATAs genes from Arabidopsis thaliana focused on GNC and GNL 180
(Behringer and Schwechheimer 2015) As yet no mutants have been 181
described for the remaining four LLM-domain B-GATAs and their biological 182
function remains elusive We therefore identified homozygous lines of T-DNA 183
insertion mutants for each of the six genes (Fig 1C Fig S3) and 184
subsequently analyzed these lines with regard to the effects of the respective 185
mutations on gene expression using quantitative real time PCR (qRT-PCR) 186
and semi-quantitative PCR (sqRT-PCR) (Fig S3B) The analysis of the gnc 187
allele (Salk_001778) which had already been used in several publications 188
suggested that neither the full-length transcript nor regions downstream from 189
the insertion site were transcribed (Fig 1C and Fig S3B) (Richter et al 2010) 190
(Bi et al 2005) Similarly analyses of the gnl insertion mutant (Salk_003995) 191
using primers spanning the entire transcript or flanking the T-DNA harbored in 192
the intron suggested that the GNL full-length transcript was absent (Fig 1C 193
and Fig S3B) (Bi et al 2005 Richter et al 2010) Although one of the 194
selected GATA15 insertion alleles (gata15-1 SAIL_618_B11) was annotated 195
as an in-gene insertion (wwwsignalsalkedu) we found no evidence for a 196
change of GATA15 gene expression in the homozygous insertion line (Fig 1C 197
and Fig S3B) Sequencing of the insertion site then revealed that the 198
insertion was downstream from the genersquos 3-UTR (untranslated region) 199
indicating that the GATA15 gene was intact in SAIL_618_B11 (Fig 1C and 200
Fig S3B) The analysis of a second allele (gata15-2 WiscDsLox471A10) with 201
an exon-insertion revealed a strong down-regulation of GATA15 expression in 202
both RT-PCR analyses (Fig 1C and Fig S3B) In a GATA16 allele with a T-203
DNA insertion in the genersquos 3-UTR (gata16-1 Salk_021471) transcript 204
abundance was partially reduced (Fig 1C and Fig S3B) The abundance of 205
GATA17 was also partially reduced in Salk_101994 (gata17-1) which 206
harbored a T-DNA insertion in the genes 5-UTR and strongly reduced in 207
SALK_049041 (gata17-2) which carried an insertion in the LLM-domain-208
encoding gene region (Fig 1C and Fig S3B) Finally we detected no 209
transcript in our analyses of a GATA17L exon insertion allele (gata17l-1 210
Salk_026798) when using primer pairs spanning the insertion or located 211
downstream from the insertion site (Fig 1C and Fig S3B) When analyzing 212
this allele with a primer combination upstream of the insertion we were able 213
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
8
to detect residual transcript in gata17l-1 but judged that this may not give rise 214
to a fully functional protein since the insertion was upstream of the LLM-215
domain (Fig 1C and Fig S3B) We concluded that the expression of the 216
respective GATA genes was absent or at least strongly reduced in alleles for 217
GNC (gnc) GNL (gnl) GATA15 (gata15-2) GATA17 (gata17-2) and 218
GATA17L (gata17l-1) alleles The gata16-1 and gata17-1 alleles were only 219
partially impaired in the expression of the respective genes whereas the 220
gata15-1 allele was not affected in GATA15 expression The gata15-2 and 221
gata17-2 alleles became available to us only later in this study and for 222
strategic reasons could not be included in the generation of the complex 223
mutant combinations described below Any of the phenotypes described in the 224
following sections for the more complex mutant combinations were not 225
apparent in the gata15-2 and gata17-2 single mutants We therefore exclude 226
the possibility that these genes are by themselves responsible for any of the 227
phenotypes described here for the higher order mutants 228
229
LLM-domain B-GATAs redundantly control greening We did not observe 230
any obvious defects in the gata single mutants apart from the already well-231
documented defects in greening of the gnc mutants (Bi et al 2005 Richter et 232
al 2010 Chiang et al 2012) To generate higher order mutants in the 233
closely related B-GATA genes we combined the alleles gnc gnl gata15-1 234
gata16-1 gata17-1 and gata17l-1 For simplicity we will subsequently omit 235
the respective allele numbers Since the presence of the misannotated 236
gata15-1 mutation does not provide information about the loss of GATA15 we 237
refer to this allele as GATA15 238
Through genetic crosses we obtained gata17 gata17l double mutants 239
GATA15 gata16 gata17 gata17l gnc gnl GATA15 gata16 and gnc gnl gata17 240
gata17l triple and quadruple mutants as well as a gnc gnl GATA15 gata16 241
gata17 gata17l quintuple mutant In the following we will refer to the gata 242
quintuple mutant as the quintuple mutant and to the GATA15 gata16 gata17 243
gata17l triple mutant that does not include the gnc and gnl alleles as the 244
(complementary) triple mutant In all other cases we will specify the allele 245
combinations 246
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
9
To examine a possible redundant function among the B-GATAs in the control 247
of greening we analyzed greening and chlorophyll content in 14 day-old light-248
grown gata mutant plants (Fig 1D and E) Although there was no indication 249
for a defect in greening in gata17 gata17l or in the triple mutant we found that 250
the greening defect of gnc gnl was further enhanced in the gnc gnl gata17 251
gata17l quadruple mutant and even more in the quintuple mutant (Fig 1D and 252
E) At the same time greening defects were not obvious in any of the gata 253
single mutants (Fig 1E) In the case of the gata15 and gata17 alleles this 254
was not due to the fact that weak alleles had been chosen for the complex 255
mutants since also the severe alleles gata15-2 and gata17-2 showed no 256
reduction in chlorophyll levels when compared to the wild type (Fig 1E) 257
The further reduction in chlorophyll accumulation in the quintuple mutant was 258
also apparent when we analyzed chlorophyll abundance by fluorescence 259
microscopy where we found reduced chloroplast fluorescence when 260
comparing the quintuple mutant to gnc gnl (Fig 1F) Furthermore we noted 261
that the greening defect gradually disappeared in gnc gnl mutants after the 262
seedling stage but remained apparent in the quintuple mutants also in adult 263
plants (Fig S4) Taken together this suggested that all mutated GATA genes 264
participate in the control of greening and chlorophyll accumulation and that 265
the defects of mutants defective in a single gene may be suppressed by the 266
presence of the other family members 267
268
Gene expression regulation of the LLM-domain B-GATAs GNC and GNL 269
had been in the focus of several studies based on the strong regulation of 270
their gene expression by light and CK (Bi et al 2005 Naito et al 2007 271
Richter et al 2010 Richter et al 2013) We were therefore interested in 272
evaluating to what extent light and CK regulate the expression of the other 273
GATA genes In the case of light regulation we found that far-red red as well 274
as blue light strongly induced the expression of GNL and to some extent also 275
the expression of GNC (Fig 2) In blue light we noted a subtle but significant 276
induction of GATA15 and GATA16 (Fig 2C) Importantly this regulation was 277
not observed in the red and far-red light receptor mutant phyA phyB or in the 278
blue light receptor mutant cry1 cry2 (Fig 2) Furthermore light-induced gene 279
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
10
expression changes in red light were impaired in the quadruple mutant for the 280
PHYTOCHROME INTERACTING FACTOR (PIF) genes PIF1 PIF3 PIF4 281
and PIF5 (pifq) indicating that the light-dependent regulation in red light was 282
mediated by the phytochrome and PIF signaling pathways (Fig S5) (Leivar et 283
al 2008) 284
The CK experiments indicated that CK induces the expression of all six GATA 285
genes (Fig 3) With an about three-fold increase in transcript abundance this 286
induction was strongest for GNL but could be observed for each of the other 287
GATA genes after a one to four hour CK treatment (Fig 3) Thus CK 288
promotes the expression of all six LLM-domain B-GATAs 289
290
Mutants of LLM-domain B-GATAs are impaired in hypocotyl elongation 291
Due to the strong regulation particularly of GNL but also of GNC in far-red red 292
and blue light conditions we examined the contribution of the GATA genes to 293
hypocotyl elongation by analyzing mutant seedlings in the different light 294
conditions in two different light intensities (Fig 4) Here we found that 295
quintuple mutant seedlings had a shorter hypocotyl than wild type seedlings in 296
white light as well as in red far-red and blue light (Fig 4) Notably these 297
phenotypes were not apparent or at least not significant in the gnc gnl 298
double mutant or the triple mutant suggesting that the respective other GATA 299
genes may contribute to the hypocotyl elongation phenotype in these 300
backgrounds (Fig 4) At the same time hypocotyl length of dark-grown 301
mutant seedlings was indistinguishable in all genotypes from that observed in 302
the wild type (Fig 4E and G) The short hypocotyl phenotype of the GATA 303
gene mutants was also interesting because we previously observed that the 304
LLM-domain B-GATA overexpressors had a longer hypocotyl than the wild 305
type when grown in white light (Behringer et al 2014) When analyzed in the 306
different light conditions we found the long hypocotyl phenotype of the GNL 307
overexpression line (GNLox) was also present in far-red and blue light but 308
interestingly not in red light where hypocotyls were even shorter than in the 309
wild type or the mutants when grown in weak red light conditions (Fig 4) 310
311
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
11
GATA gene mutants have differential gene expression defects The 312
strong induction of GNL gene expression by CK was already repeatedly 313
described and is the reason for the genersquos original designation CGA1 314
(CYTOKININ-INDUCED GATA1) (Naito et al 2007 Kollmer et al 2011) In 315
order to examine the role of the GATA genes in CK-responsive gene 316
expression we performed microarray experiments with 14 day-old wild type 317
plants gnc gnl and triple mutants as well as quintuple mutants treated for 60 318
min with the CK 6-BA (6-benzylaminopurine) or a corresponding mock solvent 319
control (Fig 5 and Fig 6 Supplemental Tables S1 and S2) We then 320
analyzed the resulting data with regard to differences in basal gene 321
expression (Fig 5) and with regard to gene expression differences after the 322
CK treatment in comparison to the mock control (Fig 6) For technical 323
reasons the experiment was conducted in two rounds the first experiment 324
included the Col-0 (1) wild type control the gnc gnl double mutant and the 325
quintuple mutant the second experiment included an independent Col-0 (2) 326
wild type control and the triple mutant When we examined the expression of 327
the CK-induced ARRs (A-TYPE RESPONSE REGULATORS) of the CK 328
signaling pathway we could confirm that the respective CK induction 329
experiments were comparably efficient between the two experiments and in 330
all genotypes tested (Fig S6A) We also examined the microarray dataset 331
with regard to the expression of a previously defined so-called ldquogolden listrdquo of 332
CK-regulated genes (Bhargava et al 2013) Over 70 of the 331 entities 333
derived from this list were significantly CK-regulated in our experiments 334
(Supplemental Table S3) When applying a two-fold expression change as 335
additional filter criterion 71 of these 331 entities were identified as up- (Col-0 336
1 and Col-0 2) and 39 (Col-0 1) and 25 entities (Col-0 2) as down-regulated 337
after CK treatment (Fig S6B and Supplemental Table S3) We thus 338
concluded that the CK treatments in both experiments were similarly efficient 339
and that the data sets could be used for comparative analyses 340
The comparison of the basal gene expression profiles of the wild type 341
samples and the mutants indicated that the (untreated) quintuple mutant 342
seedlings had by far the largest number of differentially regulated genes with 343
4722 down-regulated and 976 up-regulated entities when compared to the 344
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
12
Col-0 (1) wild type (Fig 5 and Supplemental Table S1) Furthermore the gnc 345
gnl double mutant contained 2458 down- and 294 up-regulated entities 346
respectively (Fig 5 and Supplemental Table S1) In comparison the number 347
of differentially expressed genes was comparatively minor in the triple mutant 348
which had only 323 down- and 278 up-regulated entities in this particular 349
experiment and selected physiological growth conditions (Fig 5) 350
The comparatively strong gene expression differences in the gnc gnl double 351
and the quintuple mutants and the fact that both mutants shared the gnc gnl 352
loss-of-function at the genetic level was reflected by the large overlap 353
between the differentially expressed gene sets of the two genotypes 354
Specifically 1922 of the 2458 down-regulated entities from gnc gnl were also 355
down-regulated in the quintuple mutant (Fig 5B) and 98 of the 294 entities 356
up-regulated in gnc gnl were also up-regulated in the quintuple mutant (Fig 357
5B) Conversely the overlap in gene expression defects between the triple 358
mutant and the quintuple mutant was higher than that between the triple 359
mutant and gnc gnl (Fig 5B) We therefore concluded that the gene 360
expression differences between the different GATA gene mutants was 361
strongest in the quintuple mutant and that the strong increase in the number 362
of differentially regulated genes exceeded by far the differences seen in the 363
gnc gnl double mutant and the triple mutant This supported our conclusion 364
from the phenotypic analyses that the GATA genes act in a functionally 365
redundant manner 366
Since we had observed increased gene expression of all six GATA genes 367
after CK-treatment we expected that CK-induced gene expression may be 368
compromised in the gata mutants (Fig 6) Indeed of the 996 entities that 369
were down-regulated after CK-treatment in the wild type (Col-0 1) only 173 370
(18 ) and 89 (9 ) remained CK-regulated in the quintuple and gnc gnl 371
mutant respectively (Fig 6A and Supplemental Table S2) At the same time 372
of the 188 entities that were down-regulated after CK treatment in the wild 373
type sample of the second experiment (Col-0 2) 83 (44 ) remained CK-374
regulated in the gata triple mutant (Fig 6B) The differential effect on gene 375
expression between the different mutants was less pronounced when 376
examining the CK-induced genes Here 49 (quintuple mutant) 59 (gnc 377
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
13
gnl) and 63 (triple mutant) of the CK-induced entities from the respective 378
wild types remained CK-regulated in the mutants (Fig 6A and B) In summary 379
we concluded that CK-regulated gene expression is impaired in the GATA 380
gene mutants 381
382
GATA gene mutants are defective in CK-regulated developmental 383
processes Based on the strong defects of gata mutants in CK-regulated 384
gene expression we searched for phenotypes that could be explained by 385
defects in CK response CK signaling was described as a regulator of 386
phyllotactic patterning in the shoot apical meristem of Arabidopsis (Besnard et 387
al 2014) In line with a role of the LLM-domain B-GATA genes in this process 388
we observed frequent aberrations from normal phyllotactic patterning in flower 389
positioning in triple and quintuple mutant inflorescences (Fig 7) Quantitative 390
analyses revealed aberrations from the canonical angle of ~1375deg in about 391
~30 of the petioles in triple and quintuple inflorescences (Fig 7D) At the 392
same time this patterning defect was not apparent in the gnc gnl double 393
mutant Since we also did not observe a quantitative increase in the severity 394
of this phenotype between the triple and the quintuple mutant we concluded 395
that GATA16 GATA17 and GATA17L may be responsible for this phenotype 396
(Fig 7D) 397
Leaf senescence is another CK-regulated process (Hwang et al 2012) In the 398
wild type CK levels are reduced during senescence and positive or negative 399
interference with the decrease in CK levels can promote and delay 400
senescence respectively (Hwang et al 2012) Senescence can be induced 401
by shifting light-grown plants to the dark and we had previously already shown 402
that leaf senescence is delayed in overexpression lines of LLM-domain B-403
GATAs (Behringer et al 2014) To examine possible senescence defects in 404
the gata mutants we floated leaves of wild type and mutant plants in the dark 405
on a mock solution or on a solution containing 005 microM or 01 microM 6-BA (Fig 406
8A ndash F) (Weaver and Amasino 2001) In line with the expectation that the 407
presence of CK in the medium would suppress the senescence process we 408
observed decreased senescence in CK-treated wild type leaves which we 409
quantitatively assessed by measuring chlorophyll levels in the mock- and CK-410
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
14
treated leaves (Fig 8F) Here we found that the effects of the senescence 411
treatment were less efficiently suppressed by CK treatments in the gnc gnl 412
double and in the quintuple mutant than in the wild type or the triple mutant 413
(Fig 8A - F) Similarly we observed a stronger effect on the decreases in 414
chlorophyll and total protein levels in the gnc gnl as well as the quintuple 415
mutant when we transferred light-grown plants for four days to the dark (Fig 416
8G ndash I) As previously reported GNL overexpressing plants (GNLox) had 417
senescence phenotypes that were antagonistic to the ones observed with the 418
mutants (Fig 8) (Behringer et al 2014) In the case of the leaf floating assay 419
CK-treatment had a stronger effect on greening and chlorophyll abundance in 420
GNLox than in the wild type (Fig 8A ndash F) When we transferred plants from 421
the light to the dark GNLox seedlings remained visibly green and did not 422
display the strong decrease in total protein abundance that we observed in 423
the wild type or the GATA gene mutants (Fig 8G ndash I) We thus concluded that 424
GATA gene mutants and overexpressors have antagonistic effects with 425
regard to the regulation of senescence and that GNC and GNL contribute 426
particularly strongly to this phenotype 427
CK induces greening in light-grown seedlings (Kobayashi et al 2012) To 428
examine to what extent CK can induce chlorophyll accumulation in the gata 429
mutants we grew plants for two weeks in constant light on medium containing 430
CK (5 nM BA) or a corresponding CK-free medium As expected chlorophyll 431
content was increased in wild type plants but this increase was neither 432
significant in the gnc gnl nor the triple or the quintuple mutants (Fig S7) We 433
thus concluded that the GATA factors are required for CK-induced greening in 434
Arabidopsis seedlings 435
It was previously reported that hypocotyl elongation could be repressed by 436
incubating dark-grown seedlings in the presence of CK (Su and Howell 1995) 437
Since the GATA gene mutants had displayed a hypocotyl elongation defect in 438
light-grown seedlings (Fig 4) we also explored the link between hypocotyl 439
elongation and CK However when we tested the effects of CK on hypocotyl 440
elongation in dark-grown seedlings we did not detect any significant 441
differences in the response between the different genotypes tested (Fig S8) 442
It is thus unlikely that the GATA genes participate in this elongation response 443
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
15
Branching is another phenotype that has been attributed to defects in CK 444
biosynthesis and response (El-Showk et al 2013) When we analyzed the 445
general habitus of the quintuple mutants we noted that they had an increased 446
number of secondary branches that was combined with a reduced plant 447
height (Fig 9A - C) Occasionally we also observed the outgrowth of 448
inflorescences in the axils of lateral inflorescences in the mutants whereas we 449
had not noted this phenotype in the wild type (Fig 9D and E) A quantitative 450
analysis of these phenotypes over all gata mutant phenotypes revealed that 451
the number of first and second order lateral branches increased with 452
increasing mutant complexity (Fig 9B) Here we observed strong effects 453
already in the gata17 gata17l mutant as well as the gnc gnl double mutant but 454
strongest defects in the triple and the quintuple mutant thus all five GATA 455
genes may contribute to this phenotype (Fig 9B) At the same time plant 456
height was reduced to a significant but still minor level in these mutants 457
suggesting that decreases in plant height which may have indirect 458
consequences on the general plant habitus and branching are not the 459
primary cause for altered branching patterns in the gata mutants (Fig 9C) It 460
is further important to note that the higher order GATA gene mutants initiated 461
fewer vegetative buds but had a higher number of growing buds as reflected 462
by the increased branch numbers (Fig 9F) Thus axillary bud formation is 463
promoted and bud outgrowth is repressed by the GATAs in the wild type 464
Another phenotype that became apparent during our analysis of the gata 465
mutant plant habitus was an increase in the angle formed between the 466
primary inflorescence and the lateral inflorescences (Fig 9G and H Fig S9) 467
gata mutants had a decreased angle formed between the primary 468
inflorescence and lateral inflorescences and this phenotype was strongest in 469
the quintuple mutant and intermediate in the gnc gnl and triple mutants (Fig 470
9G and H Fig S9) Interestingly we had previously observed increased 471
branch angles in GATA overexpression lines such as GNLox (Fig S9) 472
(Behringer et al 2014) We thus concluded that GATA gene mutants and 473
overexpressors antagonistically regulate branch angles in Arabidopsis 474
475
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
7
GATAs genes from Arabidopsis thaliana focused on GNC and GNL 180
(Behringer and Schwechheimer 2015) As yet no mutants have been 181
described for the remaining four LLM-domain B-GATAs and their biological 182
function remains elusive We therefore identified homozygous lines of T-DNA 183
insertion mutants for each of the six genes (Fig 1C Fig S3) and 184
subsequently analyzed these lines with regard to the effects of the respective 185
mutations on gene expression using quantitative real time PCR (qRT-PCR) 186
and semi-quantitative PCR (sqRT-PCR) (Fig S3B) The analysis of the gnc 187
allele (Salk_001778) which had already been used in several publications 188
suggested that neither the full-length transcript nor regions downstream from 189
the insertion site were transcribed (Fig 1C and Fig S3B) (Richter et al 2010) 190
(Bi et al 2005) Similarly analyses of the gnl insertion mutant (Salk_003995) 191
using primers spanning the entire transcript or flanking the T-DNA harbored in 192
the intron suggested that the GNL full-length transcript was absent (Fig 1C 193
and Fig S3B) (Bi et al 2005 Richter et al 2010) Although one of the 194
selected GATA15 insertion alleles (gata15-1 SAIL_618_B11) was annotated 195
as an in-gene insertion (wwwsignalsalkedu) we found no evidence for a 196
change of GATA15 gene expression in the homozygous insertion line (Fig 1C 197
and Fig S3B) Sequencing of the insertion site then revealed that the 198
insertion was downstream from the genersquos 3-UTR (untranslated region) 199
indicating that the GATA15 gene was intact in SAIL_618_B11 (Fig 1C and 200
Fig S3B) The analysis of a second allele (gata15-2 WiscDsLox471A10) with 201
an exon-insertion revealed a strong down-regulation of GATA15 expression in 202
both RT-PCR analyses (Fig 1C and Fig S3B) In a GATA16 allele with a T-203
DNA insertion in the genersquos 3-UTR (gata16-1 Salk_021471) transcript 204
abundance was partially reduced (Fig 1C and Fig S3B) The abundance of 205
GATA17 was also partially reduced in Salk_101994 (gata17-1) which 206
harbored a T-DNA insertion in the genes 5-UTR and strongly reduced in 207
SALK_049041 (gata17-2) which carried an insertion in the LLM-domain-208
encoding gene region (Fig 1C and Fig S3B) Finally we detected no 209
transcript in our analyses of a GATA17L exon insertion allele (gata17l-1 210
Salk_026798) when using primer pairs spanning the insertion or located 211
downstream from the insertion site (Fig 1C and Fig S3B) When analyzing 212
this allele with a primer combination upstream of the insertion we were able 213
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
8
to detect residual transcript in gata17l-1 but judged that this may not give rise 214
to a fully functional protein since the insertion was upstream of the LLM-215
domain (Fig 1C and Fig S3B) We concluded that the expression of the 216
respective GATA genes was absent or at least strongly reduced in alleles for 217
GNC (gnc) GNL (gnl) GATA15 (gata15-2) GATA17 (gata17-2) and 218
GATA17L (gata17l-1) alleles The gata16-1 and gata17-1 alleles were only 219
partially impaired in the expression of the respective genes whereas the 220
gata15-1 allele was not affected in GATA15 expression The gata15-2 and 221
gata17-2 alleles became available to us only later in this study and for 222
strategic reasons could not be included in the generation of the complex 223
mutant combinations described below Any of the phenotypes described in the 224
following sections for the more complex mutant combinations were not 225
apparent in the gata15-2 and gata17-2 single mutants We therefore exclude 226
the possibility that these genes are by themselves responsible for any of the 227
phenotypes described here for the higher order mutants 228
229
LLM-domain B-GATAs redundantly control greening We did not observe 230
any obvious defects in the gata single mutants apart from the already well-231
documented defects in greening of the gnc mutants (Bi et al 2005 Richter et 232
al 2010 Chiang et al 2012) To generate higher order mutants in the 233
closely related B-GATA genes we combined the alleles gnc gnl gata15-1 234
gata16-1 gata17-1 and gata17l-1 For simplicity we will subsequently omit 235
the respective allele numbers Since the presence of the misannotated 236
gata15-1 mutation does not provide information about the loss of GATA15 we 237
refer to this allele as GATA15 238
Through genetic crosses we obtained gata17 gata17l double mutants 239
GATA15 gata16 gata17 gata17l gnc gnl GATA15 gata16 and gnc gnl gata17 240
gata17l triple and quadruple mutants as well as a gnc gnl GATA15 gata16 241
gata17 gata17l quintuple mutant In the following we will refer to the gata 242
quintuple mutant as the quintuple mutant and to the GATA15 gata16 gata17 243
gata17l triple mutant that does not include the gnc and gnl alleles as the 244
(complementary) triple mutant In all other cases we will specify the allele 245
combinations 246
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
9
To examine a possible redundant function among the B-GATAs in the control 247
of greening we analyzed greening and chlorophyll content in 14 day-old light-248
grown gata mutant plants (Fig 1D and E) Although there was no indication 249
for a defect in greening in gata17 gata17l or in the triple mutant we found that 250
the greening defect of gnc gnl was further enhanced in the gnc gnl gata17 251
gata17l quadruple mutant and even more in the quintuple mutant (Fig 1D and 252
E) At the same time greening defects were not obvious in any of the gata 253
single mutants (Fig 1E) In the case of the gata15 and gata17 alleles this 254
was not due to the fact that weak alleles had been chosen for the complex 255
mutants since also the severe alleles gata15-2 and gata17-2 showed no 256
reduction in chlorophyll levels when compared to the wild type (Fig 1E) 257
The further reduction in chlorophyll accumulation in the quintuple mutant was 258
also apparent when we analyzed chlorophyll abundance by fluorescence 259
microscopy where we found reduced chloroplast fluorescence when 260
comparing the quintuple mutant to gnc gnl (Fig 1F) Furthermore we noted 261
that the greening defect gradually disappeared in gnc gnl mutants after the 262
seedling stage but remained apparent in the quintuple mutants also in adult 263
plants (Fig S4) Taken together this suggested that all mutated GATA genes 264
participate in the control of greening and chlorophyll accumulation and that 265
the defects of mutants defective in a single gene may be suppressed by the 266
presence of the other family members 267
268
Gene expression regulation of the LLM-domain B-GATAs GNC and GNL 269
had been in the focus of several studies based on the strong regulation of 270
their gene expression by light and CK (Bi et al 2005 Naito et al 2007 271
Richter et al 2010 Richter et al 2013) We were therefore interested in 272
evaluating to what extent light and CK regulate the expression of the other 273
GATA genes In the case of light regulation we found that far-red red as well 274
as blue light strongly induced the expression of GNL and to some extent also 275
the expression of GNC (Fig 2) In blue light we noted a subtle but significant 276
induction of GATA15 and GATA16 (Fig 2C) Importantly this regulation was 277
not observed in the red and far-red light receptor mutant phyA phyB or in the 278
blue light receptor mutant cry1 cry2 (Fig 2) Furthermore light-induced gene 279
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
10
expression changes in red light were impaired in the quadruple mutant for the 280
PHYTOCHROME INTERACTING FACTOR (PIF) genes PIF1 PIF3 PIF4 281
and PIF5 (pifq) indicating that the light-dependent regulation in red light was 282
mediated by the phytochrome and PIF signaling pathways (Fig S5) (Leivar et 283
al 2008) 284
The CK experiments indicated that CK induces the expression of all six GATA 285
genes (Fig 3) With an about three-fold increase in transcript abundance this 286
induction was strongest for GNL but could be observed for each of the other 287
GATA genes after a one to four hour CK treatment (Fig 3) Thus CK 288
promotes the expression of all six LLM-domain B-GATAs 289
290
Mutants of LLM-domain B-GATAs are impaired in hypocotyl elongation 291
Due to the strong regulation particularly of GNL but also of GNC in far-red red 292
and blue light conditions we examined the contribution of the GATA genes to 293
hypocotyl elongation by analyzing mutant seedlings in the different light 294
conditions in two different light intensities (Fig 4) Here we found that 295
quintuple mutant seedlings had a shorter hypocotyl than wild type seedlings in 296
white light as well as in red far-red and blue light (Fig 4) Notably these 297
phenotypes were not apparent or at least not significant in the gnc gnl 298
double mutant or the triple mutant suggesting that the respective other GATA 299
genes may contribute to the hypocotyl elongation phenotype in these 300
backgrounds (Fig 4) At the same time hypocotyl length of dark-grown 301
mutant seedlings was indistinguishable in all genotypes from that observed in 302
the wild type (Fig 4E and G) The short hypocotyl phenotype of the GATA 303
gene mutants was also interesting because we previously observed that the 304
LLM-domain B-GATA overexpressors had a longer hypocotyl than the wild 305
type when grown in white light (Behringer et al 2014) When analyzed in the 306
different light conditions we found the long hypocotyl phenotype of the GNL 307
overexpression line (GNLox) was also present in far-red and blue light but 308
interestingly not in red light where hypocotyls were even shorter than in the 309
wild type or the mutants when grown in weak red light conditions (Fig 4) 310
311
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
11
GATA gene mutants have differential gene expression defects The 312
strong induction of GNL gene expression by CK was already repeatedly 313
described and is the reason for the genersquos original designation CGA1 314
(CYTOKININ-INDUCED GATA1) (Naito et al 2007 Kollmer et al 2011) In 315
order to examine the role of the GATA genes in CK-responsive gene 316
expression we performed microarray experiments with 14 day-old wild type 317
plants gnc gnl and triple mutants as well as quintuple mutants treated for 60 318
min with the CK 6-BA (6-benzylaminopurine) or a corresponding mock solvent 319
control (Fig 5 and Fig 6 Supplemental Tables S1 and S2) We then 320
analyzed the resulting data with regard to differences in basal gene 321
expression (Fig 5) and with regard to gene expression differences after the 322
CK treatment in comparison to the mock control (Fig 6) For technical 323
reasons the experiment was conducted in two rounds the first experiment 324
included the Col-0 (1) wild type control the gnc gnl double mutant and the 325
quintuple mutant the second experiment included an independent Col-0 (2) 326
wild type control and the triple mutant When we examined the expression of 327
the CK-induced ARRs (A-TYPE RESPONSE REGULATORS) of the CK 328
signaling pathway we could confirm that the respective CK induction 329
experiments were comparably efficient between the two experiments and in 330
all genotypes tested (Fig S6A) We also examined the microarray dataset 331
with regard to the expression of a previously defined so-called ldquogolden listrdquo of 332
CK-regulated genes (Bhargava et al 2013) Over 70 of the 331 entities 333
derived from this list were significantly CK-regulated in our experiments 334
(Supplemental Table S3) When applying a two-fold expression change as 335
additional filter criterion 71 of these 331 entities were identified as up- (Col-0 336
1 and Col-0 2) and 39 (Col-0 1) and 25 entities (Col-0 2) as down-regulated 337
after CK treatment (Fig S6B and Supplemental Table S3) We thus 338
concluded that the CK treatments in both experiments were similarly efficient 339
and that the data sets could be used for comparative analyses 340
The comparison of the basal gene expression profiles of the wild type 341
samples and the mutants indicated that the (untreated) quintuple mutant 342
seedlings had by far the largest number of differentially regulated genes with 343
4722 down-regulated and 976 up-regulated entities when compared to the 344
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
12
Col-0 (1) wild type (Fig 5 and Supplemental Table S1) Furthermore the gnc 345
gnl double mutant contained 2458 down- and 294 up-regulated entities 346
respectively (Fig 5 and Supplemental Table S1) In comparison the number 347
of differentially expressed genes was comparatively minor in the triple mutant 348
which had only 323 down- and 278 up-regulated entities in this particular 349
experiment and selected physiological growth conditions (Fig 5) 350
The comparatively strong gene expression differences in the gnc gnl double 351
and the quintuple mutants and the fact that both mutants shared the gnc gnl 352
loss-of-function at the genetic level was reflected by the large overlap 353
between the differentially expressed gene sets of the two genotypes 354
Specifically 1922 of the 2458 down-regulated entities from gnc gnl were also 355
down-regulated in the quintuple mutant (Fig 5B) and 98 of the 294 entities 356
up-regulated in gnc gnl were also up-regulated in the quintuple mutant (Fig 357
5B) Conversely the overlap in gene expression defects between the triple 358
mutant and the quintuple mutant was higher than that between the triple 359
mutant and gnc gnl (Fig 5B) We therefore concluded that the gene 360
expression differences between the different GATA gene mutants was 361
strongest in the quintuple mutant and that the strong increase in the number 362
of differentially regulated genes exceeded by far the differences seen in the 363
gnc gnl double mutant and the triple mutant This supported our conclusion 364
from the phenotypic analyses that the GATA genes act in a functionally 365
redundant manner 366
Since we had observed increased gene expression of all six GATA genes 367
after CK-treatment we expected that CK-induced gene expression may be 368
compromised in the gata mutants (Fig 6) Indeed of the 996 entities that 369
were down-regulated after CK-treatment in the wild type (Col-0 1) only 173 370
(18 ) and 89 (9 ) remained CK-regulated in the quintuple and gnc gnl 371
mutant respectively (Fig 6A and Supplemental Table S2) At the same time 372
of the 188 entities that were down-regulated after CK treatment in the wild 373
type sample of the second experiment (Col-0 2) 83 (44 ) remained CK-374
regulated in the gata triple mutant (Fig 6B) The differential effect on gene 375
expression between the different mutants was less pronounced when 376
examining the CK-induced genes Here 49 (quintuple mutant) 59 (gnc 377
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
13
gnl) and 63 (triple mutant) of the CK-induced entities from the respective 378
wild types remained CK-regulated in the mutants (Fig 6A and B) In summary 379
we concluded that CK-regulated gene expression is impaired in the GATA 380
gene mutants 381
382
GATA gene mutants are defective in CK-regulated developmental 383
processes Based on the strong defects of gata mutants in CK-regulated 384
gene expression we searched for phenotypes that could be explained by 385
defects in CK response CK signaling was described as a regulator of 386
phyllotactic patterning in the shoot apical meristem of Arabidopsis (Besnard et 387
al 2014) In line with a role of the LLM-domain B-GATA genes in this process 388
we observed frequent aberrations from normal phyllotactic patterning in flower 389
positioning in triple and quintuple mutant inflorescences (Fig 7) Quantitative 390
analyses revealed aberrations from the canonical angle of ~1375deg in about 391
~30 of the petioles in triple and quintuple inflorescences (Fig 7D) At the 392
same time this patterning defect was not apparent in the gnc gnl double 393
mutant Since we also did not observe a quantitative increase in the severity 394
of this phenotype between the triple and the quintuple mutant we concluded 395
that GATA16 GATA17 and GATA17L may be responsible for this phenotype 396
(Fig 7D) 397
Leaf senescence is another CK-regulated process (Hwang et al 2012) In the 398
wild type CK levels are reduced during senescence and positive or negative 399
interference with the decrease in CK levels can promote and delay 400
senescence respectively (Hwang et al 2012) Senescence can be induced 401
by shifting light-grown plants to the dark and we had previously already shown 402
that leaf senescence is delayed in overexpression lines of LLM-domain B-403
GATAs (Behringer et al 2014) To examine possible senescence defects in 404
the gata mutants we floated leaves of wild type and mutant plants in the dark 405
on a mock solution or on a solution containing 005 microM or 01 microM 6-BA (Fig 406
8A ndash F) (Weaver and Amasino 2001) In line with the expectation that the 407
presence of CK in the medium would suppress the senescence process we 408
observed decreased senescence in CK-treated wild type leaves which we 409
quantitatively assessed by measuring chlorophyll levels in the mock- and CK-410
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
14
treated leaves (Fig 8F) Here we found that the effects of the senescence 411
treatment were less efficiently suppressed by CK treatments in the gnc gnl 412
double and in the quintuple mutant than in the wild type or the triple mutant 413
(Fig 8A - F) Similarly we observed a stronger effect on the decreases in 414
chlorophyll and total protein levels in the gnc gnl as well as the quintuple 415
mutant when we transferred light-grown plants for four days to the dark (Fig 416
8G ndash I) As previously reported GNL overexpressing plants (GNLox) had 417
senescence phenotypes that were antagonistic to the ones observed with the 418
mutants (Fig 8) (Behringer et al 2014) In the case of the leaf floating assay 419
CK-treatment had a stronger effect on greening and chlorophyll abundance in 420
GNLox than in the wild type (Fig 8A ndash F) When we transferred plants from 421
the light to the dark GNLox seedlings remained visibly green and did not 422
display the strong decrease in total protein abundance that we observed in 423
the wild type or the GATA gene mutants (Fig 8G ndash I) We thus concluded that 424
GATA gene mutants and overexpressors have antagonistic effects with 425
regard to the regulation of senescence and that GNC and GNL contribute 426
particularly strongly to this phenotype 427
CK induces greening in light-grown seedlings (Kobayashi et al 2012) To 428
examine to what extent CK can induce chlorophyll accumulation in the gata 429
mutants we grew plants for two weeks in constant light on medium containing 430
CK (5 nM BA) or a corresponding CK-free medium As expected chlorophyll 431
content was increased in wild type plants but this increase was neither 432
significant in the gnc gnl nor the triple or the quintuple mutants (Fig S7) We 433
thus concluded that the GATA factors are required for CK-induced greening in 434
Arabidopsis seedlings 435
It was previously reported that hypocotyl elongation could be repressed by 436
incubating dark-grown seedlings in the presence of CK (Su and Howell 1995) 437
Since the GATA gene mutants had displayed a hypocotyl elongation defect in 438
light-grown seedlings (Fig 4) we also explored the link between hypocotyl 439
elongation and CK However when we tested the effects of CK on hypocotyl 440
elongation in dark-grown seedlings we did not detect any significant 441
differences in the response between the different genotypes tested (Fig S8) 442
It is thus unlikely that the GATA genes participate in this elongation response 443
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
15
Branching is another phenotype that has been attributed to defects in CK 444
biosynthesis and response (El-Showk et al 2013) When we analyzed the 445
general habitus of the quintuple mutants we noted that they had an increased 446
number of secondary branches that was combined with a reduced plant 447
height (Fig 9A - C) Occasionally we also observed the outgrowth of 448
inflorescences in the axils of lateral inflorescences in the mutants whereas we 449
had not noted this phenotype in the wild type (Fig 9D and E) A quantitative 450
analysis of these phenotypes over all gata mutant phenotypes revealed that 451
the number of first and second order lateral branches increased with 452
increasing mutant complexity (Fig 9B) Here we observed strong effects 453
already in the gata17 gata17l mutant as well as the gnc gnl double mutant but 454
strongest defects in the triple and the quintuple mutant thus all five GATA 455
genes may contribute to this phenotype (Fig 9B) At the same time plant 456
height was reduced to a significant but still minor level in these mutants 457
suggesting that decreases in plant height which may have indirect 458
consequences on the general plant habitus and branching are not the 459
primary cause for altered branching patterns in the gata mutants (Fig 9C) It 460
is further important to note that the higher order GATA gene mutants initiated 461
fewer vegetative buds but had a higher number of growing buds as reflected 462
by the increased branch numbers (Fig 9F) Thus axillary bud formation is 463
promoted and bud outgrowth is repressed by the GATAs in the wild type 464
Another phenotype that became apparent during our analysis of the gata 465
mutant plant habitus was an increase in the angle formed between the 466
primary inflorescence and the lateral inflorescences (Fig 9G and H Fig S9) 467
gata mutants had a decreased angle formed between the primary 468
inflorescence and lateral inflorescences and this phenotype was strongest in 469
the quintuple mutant and intermediate in the gnc gnl and triple mutants (Fig 470
9G and H Fig S9) Interestingly we had previously observed increased 471
branch angles in GATA overexpression lines such as GNLox (Fig S9) 472
(Behringer et al 2014) We thus concluded that GATA gene mutants and 473
overexpressors antagonistically regulate branch angles in Arabidopsis 474
475
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
8
to detect residual transcript in gata17l-1 but judged that this may not give rise 214
to a fully functional protein since the insertion was upstream of the LLM-215
domain (Fig 1C and Fig S3B) We concluded that the expression of the 216
respective GATA genes was absent or at least strongly reduced in alleles for 217
GNC (gnc) GNL (gnl) GATA15 (gata15-2) GATA17 (gata17-2) and 218
GATA17L (gata17l-1) alleles The gata16-1 and gata17-1 alleles were only 219
partially impaired in the expression of the respective genes whereas the 220
gata15-1 allele was not affected in GATA15 expression The gata15-2 and 221
gata17-2 alleles became available to us only later in this study and for 222
strategic reasons could not be included in the generation of the complex 223
mutant combinations described below Any of the phenotypes described in the 224
following sections for the more complex mutant combinations were not 225
apparent in the gata15-2 and gata17-2 single mutants We therefore exclude 226
the possibility that these genes are by themselves responsible for any of the 227
phenotypes described here for the higher order mutants 228
229
LLM-domain B-GATAs redundantly control greening We did not observe 230
any obvious defects in the gata single mutants apart from the already well-231
documented defects in greening of the gnc mutants (Bi et al 2005 Richter et 232
al 2010 Chiang et al 2012) To generate higher order mutants in the 233
closely related B-GATA genes we combined the alleles gnc gnl gata15-1 234
gata16-1 gata17-1 and gata17l-1 For simplicity we will subsequently omit 235
the respective allele numbers Since the presence of the misannotated 236
gata15-1 mutation does not provide information about the loss of GATA15 we 237
refer to this allele as GATA15 238
Through genetic crosses we obtained gata17 gata17l double mutants 239
GATA15 gata16 gata17 gata17l gnc gnl GATA15 gata16 and gnc gnl gata17 240
gata17l triple and quadruple mutants as well as a gnc gnl GATA15 gata16 241
gata17 gata17l quintuple mutant In the following we will refer to the gata 242
quintuple mutant as the quintuple mutant and to the GATA15 gata16 gata17 243
gata17l triple mutant that does not include the gnc and gnl alleles as the 244
(complementary) triple mutant In all other cases we will specify the allele 245
combinations 246
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
9
To examine a possible redundant function among the B-GATAs in the control 247
of greening we analyzed greening and chlorophyll content in 14 day-old light-248
grown gata mutant plants (Fig 1D and E) Although there was no indication 249
for a defect in greening in gata17 gata17l or in the triple mutant we found that 250
the greening defect of gnc gnl was further enhanced in the gnc gnl gata17 251
gata17l quadruple mutant and even more in the quintuple mutant (Fig 1D and 252
E) At the same time greening defects were not obvious in any of the gata 253
single mutants (Fig 1E) In the case of the gata15 and gata17 alleles this 254
was not due to the fact that weak alleles had been chosen for the complex 255
mutants since also the severe alleles gata15-2 and gata17-2 showed no 256
reduction in chlorophyll levels when compared to the wild type (Fig 1E) 257
The further reduction in chlorophyll accumulation in the quintuple mutant was 258
also apparent when we analyzed chlorophyll abundance by fluorescence 259
microscopy where we found reduced chloroplast fluorescence when 260
comparing the quintuple mutant to gnc gnl (Fig 1F) Furthermore we noted 261
that the greening defect gradually disappeared in gnc gnl mutants after the 262
seedling stage but remained apparent in the quintuple mutants also in adult 263
plants (Fig S4) Taken together this suggested that all mutated GATA genes 264
participate in the control of greening and chlorophyll accumulation and that 265
the defects of mutants defective in a single gene may be suppressed by the 266
presence of the other family members 267
268
Gene expression regulation of the LLM-domain B-GATAs GNC and GNL 269
had been in the focus of several studies based on the strong regulation of 270
their gene expression by light and CK (Bi et al 2005 Naito et al 2007 271
Richter et al 2010 Richter et al 2013) We were therefore interested in 272
evaluating to what extent light and CK regulate the expression of the other 273
GATA genes In the case of light regulation we found that far-red red as well 274
as blue light strongly induced the expression of GNL and to some extent also 275
the expression of GNC (Fig 2) In blue light we noted a subtle but significant 276
induction of GATA15 and GATA16 (Fig 2C) Importantly this regulation was 277
not observed in the red and far-red light receptor mutant phyA phyB or in the 278
blue light receptor mutant cry1 cry2 (Fig 2) Furthermore light-induced gene 279
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
10
expression changes in red light were impaired in the quadruple mutant for the 280
PHYTOCHROME INTERACTING FACTOR (PIF) genes PIF1 PIF3 PIF4 281
and PIF5 (pifq) indicating that the light-dependent regulation in red light was 282
mediated by the phytochrome and PIF signaling pathways (Fig S5) (Leivar et 283
al 2008) 284
The CK experiments indicated that CK induces the expression of all six GATA 285
genes (Fig 3) With an about three-fold increase in transcript abundance this 286
induction was strongest for GNL but could be observed for each of the other 287
GATA genes after a one to four hour CK treatment (Fig 3) Thus CK 288
promotes the expression of all six LLM-domain B-GATAs 289
290
Mutants of LLM-domain B-GATAs are impaired in hypocotyl elongation 291
Due to the strong regulation particularly of GNL but also of GNC in far-red red 292
and blue light conditions we examined the contribution of the GATA genes to 293
hypocotyl elongation by analyzing mutant seedlings in the different light 294
conditions in two different light intensities (Fig 4) Here we found that 295
quintuple mutant seedlings had a shorter hypocotyl than wild type seedlings in 296
white light as well as in red far-red and blue light (Fig 4) Notably these 297
phenotypes were not apparent or at least not significant in the gnc gnl 298
double mutant or the triple mutant suggesting that the respective other GATA 299
genes may contribute to the hypocotyl elongation phenotype in these 300
backgrounds (Fig 4) At the same time hypocotyl length of dark-grown 301
mutant seedlings was indistinguishable in all genotypes from that observed in 302
the wild type (Fig 4E and G) The short hypocotyl phenotype of the GATA 303
gene mutants was also interesting because we previously observed that the 304
LLM-domain B-GATA overexpressors had a longer hypocotyl than the wild 305
type when grown in white light (Behringer et al 2014) When analyzed in the 306
different light conditions we found the long hypocotyl phenotype of the GNL 307
overexpression line (GNLox) was also present in far-red and blue light but 308
interestingly not in red light where hypocotyls were even shorter than in the 309
wild type or the mutants when grown in weak red light conditions (Fig 4) 310
311
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
11
GATA gene mutants have differential gene expression defects The 312
strong induction of GNL gene expression by CK was already repeatedly 313
described and is the reason for the genersquos original designation CGA1 314
(CYTOKININ-INDUCED GATA1) (Naito et al 2007 Kollmer et al 2011) In 315
order to examine the role of the GATA genes in CK-responsive gene 316
expression we performed microarray experiments with 14 day-old wild type 317
plants gnc gnl and triple mutants as well as quintuple mutants treated for 60 318
min with the CK 6-BA (6-benzylaminopurine) or a corresponding mock solvent 319
control (Fig 5 and Fig 6 Supplemental Tables S1 and S2) We then 320
analyzed the resulting data with regard to differences in basal gene 321
expression (Fig 5) and with regard to gene expression differences after the 322
CK treatment in comparison to the mock control (Fig 6) For technical 323
reasons the experiment was conducted in two rounds the first experiment 324
included the Col-0 (1) wild type control the gnc gnl double mutant and the 325
quintuple mutant the second experiment included an independent Col-0 (2) 326
wild type control and the triple mutant When we examined the expression of 327
the CK-induced ARRs (A-TYPE RESPONSE REGULATORS) of the CK 328
signaling pathway we could confirm that the respective CK induction 329
experiments were comparably efficient between the two experiments and in 330
all genotypes tested (Fig S6A) We also examined the microarray dataset 331
with regard to the expression of a previously defined so-called ldquogolden listrdquo of 332
CK-regulated genes (Bhargava et al 2013) Over 70 of the 331 entities 333
derived from this list were significantly CK-regulated in our experiments 334
(Supplemental Table S3) When applying a two-fold expression change as 335
additional filter criterion 71 of these 331 entities were identified as up- (Col-0 336
1 and Col-0 2) and 39 (Col-0 1) and 25 entities (Col-0 2) as down-regulated 337
after CK treatment (Fig S6B and Supplemental Table S3) We thus 338
concluded that the CK treatments in both experiments were similarly efficient 339
and that the data sets could be used for comparative analyses 340
The comparison of the basal gene expression profiles of the wild type 341
samples and the mutants indicated that the (untreated) quintuple mutant 342
seedlings had by far the largest number of differentially regulated genes with 343
4722 down-regulated and 976 up-regulated entities when compared to the 344
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
12
Col-0 (1) wild type (Fig 5 and Supplemental Table S1) Furthermore the gnc 345
gnl double mutant contained 2458 down- and 294 up-regulated entities 346
respectively (Fig 5 and Supplemental Table S1) In comparison the number 347
of differentially expressed genes was comparatively minor in the triple mutant 348
which had only 323 down- and 278 up-regulated entities in this particular 349
experiment and selected physiological growth conditions (Fig 5) 350
The comparatively strong gene expression differences in the gnc gnl double 351
and the quintuple mutants and the fact that both mutants shared the gnc gnl 352
loss-of-function at the genetic level was reflected by the large overlap 353
between the differentially expressed gene sets of the two genotypes 354
Specifically 1922 of the 2458 down-regulated entities from gnc gnl were also 355
down-regulated in the quintuple mutant (Fig 5B) and 98 of the 294 entities 356
up-regulated in gnc gnl were also up-regulated in the quintuple mutant (Fig 357
5B) Conversely the overlap in gene expression defects between the triple 358
mutant and the quintuple mutant was higher than that between the triple 359
mutant and gnc gnl (Fig 5B) We therefore concluded that the gene 360
expression differences between the different GATA gene mutants was 361
strongest in the quintuple mutant and that the strong increase in the number 362
of differentially regulated genes exceeded by far the differences seen in the 363
gnc gnl double mutant and the triple mutant This supported our conclusion 364
from the phenotypic analyses that the GATA genes act in a functionally 365
redundant manner 366
Since we had observed increased gene expression of all six GATA genes 367
after CK-treatment we expected that CK-induced gene expression may be 368
compromised in the gata mutants (Fig 6) Indeed of the 996 entities that 369
were down-regulated after CK-treatment in the wild type (Col-0 1) only 173 370
(18 ) and 89 (9 ) remained CK-regulated in the quintuple and gnc gnl 371
mutant respectively (Fig 6A and Supplemental Table S2) At the same time 372
of the 188 entities that were down-regulated after CK treatment in the wild 373
type sample of the second experiment (Col-0 2) 83 (44 ) remained CK-374
regulated in the gata triple mutant (Fig 6B) The differential effect on gene 375
expression between the different mutants was less pronounced when 376
examining the CK-induced genes Here 49 (quintuple mutant) 59 (gnc 377
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
13
gnl) and 63 (triple mutant) of the CK-induced entities from the respective 378
wild types remained CK-regulated in the mutants (Fig 6A and B) In summary 379
we concluded that CK-regulated gene expression is impaired in the GATA 380
gene mutants 381
382
GATA gene mutants are defective in CK-regulated developmental 383
processes Based on the strong defects of gata mutants in CK-regulated 384
gene expression we searched for phenotypes that could be explained by 385
defects in CK response CK signaling was described as a regulator of 386
phyllotactic patterning in the shoot apical meristem of Arabidopsis (Besnard et 387
al 2014) In line with a role of the LLM-domain B-GATA genes in this process 388
we observed frequent aberrations from normal phyllotactic patterning in flower 389
positioning in triple and quintuple mutant inflorescences (Fig 7) Quantitative 390
analyses revealed aberrations from the canonical angle of ~1375deg in about 391
~30 of the petioles in triple and quintuple inflorescences (Fig 7D) At the 392
same time this patterning defect was not apparent in the gnc gnl double 393
mutant Since we also did not observe a quantitative increase in the severity 394
of this phenotype between the triple and the quintuple mutant we concluded 395
that GATA16 GATA17 and GATA17L may be responsible for this phenotype 396
(Fig 7D) 397
Leaf senescence is another CK-regulated process (Hwang et al 2012) In the 398
wild type CK levels are reduced during senescence and positive or negative 399
interference with the decrease in CK levels can promote and delay 400
senescence respectively (Hwang et al 2012) Senescence can be induced 401
by shifting light-grown plants to the dark and we had previously already shown 402
that leaf senescence is delayed in overexpression lines of LLM-domain B-403
GATAs (Behringer et al 2014) To examine possible senescence defects in 404
the gata mutants we floated leaves of wild type and mutant plants in the dark 405
on a mock solution or on a solution containing 005 microM or 01 microM 6-BA (Fig 406
8A ndash F) (Weaver and Amasino 2001) In line with the expectation that the 407
presence of CK in the medium would suppress the senescence process we 408
observed decreased senescence in CK-treated wild type leaves which we 409
quantitatively assessed by measuring chlorophyll levels in the mock- and CK-410
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
14
treated leaves (Fig 8F) Here we found that the effects of the senescence 411
treatment were less efficiently suppressed by CK treatments in the gnc gnl 412
double and in the quintuple mutant than in the wild type or the triple mutant 413
(Fig 8A - F) Similarly we observed a stronger effect on the decreases in 414
chlorophyll and total protein levels in the gnc gnl as well as the quintuple 415
mutant when we transferred light-grown plants for four days to the dark (Fig 416
8G ndash I) As previously reported GNL overexpressing plants (GNLox) had 417
senescence phenotypes that were antagonistic to the ones observed with the 418
mutants (Fig 8) (Behringer et al 2014) In the case of the leaf floating assay 419
CK-treatment had a stronger effect on greening and chlorophyll abundance in 420
GNLox than in the wild type (Fig 8A ndash F) When we transferred plants from 421
the light to the dark GNLox seedlings remained visibly green and did not 422
display the strong decrease in total protein abundance that we observed in 423
the wild type or the GATA gene mutants (Fig 8G ndash I) We thus concluded that 424
GATA gene mutants and overexpressors have antagonistic effects with 425
regard to the regulation of senescence and that GNC and GNL contribute 426
particularly strongly to this phenotype 427
CK induces greening in light-grown seedlings (Kobayashi et al 2012) To 428
examine to what extent CK can induce chlorophyll accumulation in the gata 429
mutants we grew plants for two weeks in constant light on medium containing 430
CK (5 nM BA) or a corresponding CK-free medium As expected chlorophyll 431
content was increased in wild type plants but this increase was neither 432
significant in the gnc gnl nor the triple or the quintuple mutants (Fig S7) We 433
thus concluded that the GATA factors are required for CK-induced greening in 434
Arabidopsis seedlings 435
It was previously reported that hypocotyl elongation could be repressed by 436
incubating dark-grown seedlings in the presence of CK (Su and Howell 1995) 437
Since the GATA gene mutants had displayed a hypocotyl elongation defect in 438
light-grown seedlings (Fig 4) we also explored the link between hypocotyl 439
elongation and CK However when we tested the effects of CK on hypocotyl 440
elongation in dark-grown seedlings we did not detect any significant 441
differences in the response between the different genotypes tested (Fig S8) 442
It is thus unlikely that the GATA genes participate in this elongation response 443
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
15
Branching is another phenotype that has been attributed to defects in CK 444
biosynthesis and response (El-Showk et al 2013) When we analyzed the 445
general habitus of the quintuple mutants we noted that they had an increased 446
number of secondary branches that was combined with a reduced plant 447
height (Fig 9A - C) Occasionally we also observed the outgrowth of 448
inflorescences in the axils of lateral inflorescences in the mutants whereas we 449
had not noted this phenotype in the wild type (Fig 9D and E) A quantitative 450
analysis of these phenotypes over all gata mutant phenotypes revealed that 451
the number of first and second order lateral branches increased with 452
increasing mutant complexity (Fig 9B) Here we observed strong effects 453
already in the gata17 gata17l mutant as well as the gnc gnl double mutant but 454
strongest defects in the triple and the quintuple mutant thus all five GATA 455
genes may contribute to this phenotype (Fig 9B) At the same time plant 456
height was reduced to a significant but still minor level in these mutants 457
suggesting that decreases in plant height which may have indirect 458
consequences on the general plant habitus and branching are not the 459
primary cause for altered branching patterns in the gata mutants (Fig 9C) It 460
is further important to note that the higher order GATA gene mutants initiated 461
fewer vegetative buds but had a higher number of growing buds as reflected 462
by the increased branch numbers (Fig 9F) Thus axillary bud formation is 463
promoted and bud outgrowth is repressed by the GATAs in the wild type 464
Another phenotype that became apparent during our analysis of the gata 465
mutant plant habitus was an increase in the angle formed between the 466
primary inflorescence and the lateral inflorescences (Fig 9G and H Fig S9) 467
gata mutants had a decreased angle formed between the primary 468
inflorescence and lateral inflorescences and this phenotype was strongest in 469
the quintuple mutant and intermediate in the gnc gnl and triple mutants (Fig 470
9G and H Fig S9) Interestingly we had previously observed increased 471
branch angles in GATA overexpression lines such as GNLox (Fig S9) 472
(Behringer et al 2014) We thus concluded that GATA gene mutants and 473
overexpressors antagonistically regulate branch angles in Arabidopsis 474
475
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
9
To examine a possible redundant function among the B-GATAs in the control 247
of greening we analyzed greening and chlorophyll content in 14 day-old light-248
grown gata mutant plants (Fig 1D and E) Although there was no indication 249
for a defect in greening in gata17 gata17l or in the triple mutant we found that 250
the greening defect of gnc gnl was further enhanced in the gnc gnl gata17 251
gata17l quadruple mutant and even more in the quintuple mutant (Fig 1D and 252
E) At the same time greening defects were not obvious in any of the gata 253
single mutants (Fig 1E) In the case of the gata15 and gata17 alleles this 254
was not due to the fact that weak alleles had been chosen for the complex 255
mutants since also the severe alleles gata15-2 and gata17-2 showed no 256
reduction in chlorophyll levels when compared to the wild type (Fig 1E) 257
The further reduction in chlorophyll accumulation in the quintuple mutant was 258
also apparent when we analyzed chlorophyll abundance by fluorescence 259
microscopy where we found reduced chloroplast fluorescence when 260
comparing the quintuple mutant to gnc gnl (Fig 1F) Furthermore we noted 261
that the greening defect gradually disappeared in gnc gnl mutants after the 262
seedling stage but remained apparent in the quintuple mutants also in adult 263
plants (Fig S4) Taken together this suggested that all mutated GATA genes 264
participate in the control of greening and chlorophyll accumulation and that 265
the defects of mutants defective in a single gene may be suppressed by the 266
presence of the other family members 267
268
Gene expression regulation of the LLM-domain B-GATAs GNC and GNL 269
had been in the focus of several studies based on the strong regulation of 270
their gene expression by light and CK (Bi et al 2005 Naito et al 2007 271
Richter et al 2010 Richter et al 2013) We were therefore interested in 272
evaluating to what extent light and CK regulate the expression of the other 273
GATA genes In the case of light regulation we found that far-red red as well 274
as blue light strongly induced the expression of GNL and to some extent also 275
the expression of GNC (Fig 2) In blue light we noted a subtle but significant 276
induction of GATA15 and GATA16 (Fig 2C) Importantly this regulation was 277
not observed in the red and far-red light receptor mutant phyA phyB or in the 278
blue light receptor mutant cry1 cry2 (Fig 2) Furthermore light-induced gene 279
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
10
expression changes in red light were impaired in the quadruple mutant for the 280
PHYTOCHROME INTERACTING FACTOR (PIF) genes PIF1 PIF3 PIF4 281
and PIF5 (pifq) indicating that the light-dependent regulation in red light was 282
mediated by the phytochrome and PIF signaling pathways (Fig S5) (Leivar et 283
al 2008) 284
The CK experiments indicated that CK induces the expression of all six GATA 285
genes (Fig 3) With an about three-fold increase in transcript abundance this 286
induction was strongest for GNL but could be observed for each of the other 287
GATA genes after a one to four hour CK treatment (Fig 3) Thus CK 288
promotes the expression of all six LLM-domain B-GATAs 289
290
Mutants of LLM-domain B-GATAs are impaired in hypocotyl elongation 291
Due to the strong regulation particularly of GNL but also of GNC in far-red red 292
and blue light conditions we examined the contribution of the GATA genes to 293
hypocotyl elongation by analyzing mutant seedlings in the different light 294
conditions in two different light intensities (Fig 4) Here we found that 295
quintuple mutant seedlings had a shorter hypocotyl than wild type seedlings in 296
white light as well as in red far-red and blue light (Fig 4) Notably these 297
phenotypes were not apparent or at least not significant in the gnc gnl 298
double mutant or the triple mutant suggesting that the respective other GATA 299
genes may contribute to the hypocotyl elongation phenotype in these 300
backgrounds (Fig 4) At the same time hypocotyl length of dark-grown 301
mutant seedlings was indistinguishable in all genotypes from that observed in 302
the wild type (Fig 4E and G) The short hypocotyl phenotype of the GATA 303
gene mutants was also interesting because we previously observed that the 304
LLM-domain B-GATA overexpressors had a longer hypocotyl than the wild 305
type when grown in white light (Behringer et al 2014) When analyzed in the 306
different light conditions we found the long hypocotyl phenotype of the GNL 307
overexpression line (GNLox) was also present in far-red and blue light but 308
interestingly not in red light where hypocotyls were even shorter than in the 309
wild type or the mutants when grown in weak red light conditions (Fig 4) 310
311
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
11
GATA gene mutants have differential gene expression defects The 312
strong induction of GNL gene expression by CK was already repeatedly 313
described and is the reason for the genersquos original designation CGA1 314
(CYTOKININ-INDUCED GATA1) (Naito et al 2007 Kollmer et al 2011) In 315
order to examine the role of the GATA genes in CK-responsive gene 316
expression we performed microarray experiments with 14 day-old wild type 317
plants gnc gnl and triple mutants as well as quintuple mutants treated for 60 318
min with the CK 6-BA (6-benzylaminopurine) or a corresponding mock solvent 319
control (Fig 5 and Fig 6 Supplemental Tables S1 and S2) We then 320
analyzed the resulting data with regard to differences in basal gene 321
expression (Fig 5) and with regard to gene expression differences after the 322
CK treatment in comparison to the mock control (Fig 6) For technical 323
reasons the experiment was conducted in two rounds the first experiment 324
included the Col-0 (1) wild type control the gnc gnl double mutant and the 325
quintuple mutant the second experiment included an independent Col-0 (2) 326
wild type control and the triple mutant When we examined the expression of 327
the CK-induced ARRs (A-TYPE RESPONSE REGULATORS) of the CK 328
signaling pathway we could confirm that the respective CK induction 329
experiments were comparably efficient between the two experiments and in 330
all genotypes tested (Fig S6A) We also examined the microarray dataset 331
with regard to the expression of a previously defined so-called ldquogolden listrdquo of 332
CK-regulated genes (Bhargava et al 2013) Over 70 of the 331 entities 333
derived from this list were significantly CK-regulated in our experiments 334
(Supplemental Table S3) When applying a two-fold expression change as 335
additional filter criterion 71 of these 331 entities were identified as up- (Col-0 336
1 and Col-0 2) and 39 (Col-0 1) and 25 entities (Col-0 2) as down-regulated 337
after CK treatment (Fig S6B and Supplemental Table S3) We thus 338
concluded that the CK treatments in both experiments were similarly efficient 339
and that the data sets could be used for comparative analyses 340
The comparison of the basal gene expression profiles of the wild type 341
samples and the mutants indicated that the (untreated) quintuple mutant 342
seedlings had by far the largest number of differentially regulated genes with 343
4722 down-regulated and 976 up-regulated entities when compared to the 344
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
12
Col-0 (1) wild type (Fig 5 and Supplemental Table S1) Furthermore the gnc 345
gnl double mutant contained 2458 down- and 294 up-regulated entities 346
respectively (Fig 5 and Supplemental Table S1) In comparison the number 347
of differentially expressed genes was comparatively minor in the triple mutant 348
which had only 323 down- and 278 up-regulated entities in this particular 349
experiment and selected physiological growth conditions (Fig 5) 350
The comparatively strong gene expression differences in the gnc gnl double 351
and the quintuple mutants and the fact that both mutants shared the gnc gnl 352
loss-of-function at the genetic level was reflected by the large overlap 353
between the differentially expressed gene sets of the two genotypes 354
Specifically 1922 of the 2458 down-regulated entities from gnc gnl were also 355
down-regulated in the quintuple mutant (Fig 5B) and 98 of the 294 entities 356
up-regulated in gnc gnl were also up-regulated in the quintuple mutant (Fig 357
5B) Conversely the overlap in gene expression defects between the triple 358
mutant and the quintuple mutant was higher than that between the triple 359
mutant and gnc gnl (Fig 5B) We therefore concluded that the gene 360
expression differences between the different GATA gene mutants was 361
strongest in the quintuple mutant and that the strong increase in the number 362
of differentially regulated genes exceeded by far the differences seen in the 363
gnc gnl double mutant and the triple mutant This supported our conclusion 364
from the phenotypic analyses that the GATA genes act in a functionally 365
redundant manner 366
Since we had observed increased gene expression of all six GATA genes 367
after CK-treatment we expected that CK-induced gene expression may be 368
compromised in the gata mutants (Fig 6) Indeed of the 996 entities that 369
were down-regulated after CK-treatment in the wild type (Col-0 1) only 173 370
(18 ) and 89 (9 ) remained CK-regulated in the quintuple and gnc gnl 371
mutant respectively (Fig 6A and Supplemental Table S2) At the same time 372
of the 188 entities that were down-regulated after CK treatment in the wild 373
type sample of the second experiment (Col-0 2) 83 (44 ) remained CK-374
regulated in the gata triple mutant (Fig 6B) The differential effect on gene 375
expression between the different mutants was less pronounced when 376
examining the CK-induced genes Here 49 (quintuple mutant) 59 (gnc 377
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
13
gnl) and 63 (triple mutant) of the CK-induced entities from the respective 378
wild types remained CK-regulated in the mutants (Fig 6A and B) In summary 379
we concluded that CK-regulated gene expression is impaired in the GATA 380
gene mutants 381
382
GATA gene mutants are defective in CK-regulated developmental 383
processes Based on the strong defects of gata mutants in CK-regulated 384
gene expression we searched for phenotypes that could be explained by 385
defects in CK response CK signaling was described as a regulator of 386
phyllotactic patterning in the shoot apical meristem of Arabidopsis (Besnard et 387
al 2014) In line with a role of the LLM-domain B-GATA genes in this process 388
we observed frequent aberrations from normal phyllotactic patterning in flower 389
positioning in triple and quintuple mutant inflorescences (Fig 7) Quantitative 390
analyses revealed aberrations from the canonical angle of ~1375deg in about 391
~30 of the petioles in triple and quintuple inflorescences (Fig 7D) At the 392
same time this patterning defect was not apparent in the gnc gnl double 393
mutant Since we also did not observe a quantitative increase in the severity 394
of this phenotype between the triple and the quintuple mutant we concluded 395
that GATA16 GATA17 and GATA17L may be responsible for this phenotype 396
(Fig 7D) 397
Leaf senescence is another CK-regulated process (Hwang et al 2012) In the 398
wild type CK levels are reduced during senescence and positive or negative 399
interference with the decrease in CK levels can promote and delay 400
senescence respectively (Hwang et al 2012) Senescence can be induced 401
by shifting light-grown plants to the dark and we had previously already shown 402
that leaf senescence is delayed in overexpression lines of LLM-domain B-403
GATAs (Behringer et al 2014) To examine possible senescence defects in 404
the gata mutants we floated leaves of wild type and mutant plants in the dark 405
on a mock solution or on a solution containing 005 microM or 01 microM 6-BA (Fig 406
8A ndash F) (Weaver and Amasino 2001) In line with the expectation that the 407
presence of CK in the medium would suppress the senescence process we 408
observed decreased senescence in CK-treated wild type leaves which we 409
quantitatively assessed by measuring chlorophyll levels in the mock- and CK-410
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
14
treated leaves (Fig 8F) Here we found that the effects of the senescence 411
treatment were less efficiently suppressed by CK treatments in the gnc gnl 412
double and in the quintuple mutant than in the wild type or the triple mutant 413
(Fig 8A - F) Similarly we observed a stronger effect on the decreases in 414
chlorophyll and total protein levels in the gnc gnl as well as the quintuple 415
mutant when we transferred light-grown plants for four days to the dark (Fig 416
8G ndash I) As previously reported GNL overexpressing plants (GNLox) had 417
senescence phenotypes that were antagonistic to the ones observed with the 418
mutants (Fig 8) (Behringer et al 2014) In the case of the leaf floating assay 419
CK-treatment had a stronger effect on greening and chlorophyll abundance in 420
GNLox than in the wild type (Fig 8A ndash F) When we transferred plants from 421
the light to the dark GNLox seedlings remained visibly green and did not 422
display the strong decrease in total protein abundance that we observed in 423
the wild type or the GATA gene mutants (Fig 8G ndash I) We thus concluded that 424
GATA gene mutants and overexpressors have antagonistic effects with 425
regard to the regulation of senescence and that GNC and GNL contribute 426
particularly strongly to this phenotype 427
CK induces greening in light-grown seedlings (Kobayashi et al 2012) To 428
examine to what extent CK can induce chlorophyll accumulation in the gata 429
mutants we grew plants for two weeks in constant light on medium containing 430
CK (5 nM BA) or a corresponding CK-free medium As expected chlorophyll 431
content was increased in wild type plants but this increase was neither 432
significant in the gnc gnl nor the triple or the quintuple mutants (Fig S7) We 433
thus concluded that the GATA factors are required for CK-induced greening in 434
Arabidopsis seedlings 435
It was previously reported that hypocotyl elongation could be repressed by 436
incubating dark-grown seedlings in the presence of CK (Su and Howell 1995) 437
Since the GATA gene mutants had displayed a hypocotyl elongation defect in 438
light-grown seedlings (Fig 4) we also explored the link between hypocotyl 439
elongation and CK However when we tested the effects of CK on hypocotyl 440
elongation in dark-grown seedlings we did not detect any significant 441
differences in the response between the different genotypes tested (Fig S8) 442
It is thus unlikely that the GATA genes participate in this elongation response 443
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
15
Branching is another phenotype that has been attributed to defects in CK 444
biosynthesis and response (El-Showk et al 2013) When we analyzed the 445
general habitus of the quintuple mutants we noted that they had an increased 446
number of secondary branches that was combined with a reduced plant 447
height (Fig 9A - C) Occasionally we also observed the outgrowth of 448
inflorescences in the axils of lateral inflorescences in the mutants whereas we 449
had not noted this phenotype in the wild type (Fig 9D and E) A quantitative 450
analysis of these phenotypes over all gata mutant phenotypes revealed that 451
the number of first and second order lateral branches increased with 452
increasing mutant complexity (Fig 9B) Here we observed strong effects 453
already in the gata17 gata17l mutant as well as the gnc gnl double mutant but 454
strongest defects in the triple and the quintuple mutant thus all five GATA 455
genes may contribute to this phenotype (Fig 9B) At the same time plant 456
height was reduced to a significant but still minor level in these mutants 457
suggesting that decreases in plant height which may have indirect 458
consequences on the general plant habitus and branching are not the 459
primary cause for altered branching patterns in the gata mutants (Fig 9C) It 460
is further important to note that the higher order GATA gene mutants initiated 461
fewer vegetative buds but had a higher number of growing buds as reflected 462
by the increased branch numbers (Fig 9F) Thus axillary bud formation is 463
promoted and bud outgrowth is repressed by the GATAs in the wild type 464
Another phenotype that became apparent during our analysis of the gata 465
mutant plant habitus was an increase in the angle formed between the 466
primary inflorescence and the lateral inflorescences (Fig 9G and H Fig S9) 467
gata mutants had a decreased angle formed between the primary 468
inflorescence and lateral inflorescences and this phenotype was strongest in 469
the quintuple mutant and intermediate in the gnc gnl and triple mutants (Fig 470
9G and H Fig S9) Interestingly we had previously observed increased 471
branch angles in GATA overexpression lines such as GNLox (Fig S9) 472
(Behringer et al 2014) We thus concluded that GATA gene mutants and 473
overexpressors antagonistically regulate branch angles in Arabidopsis 474
475
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
10
expression changes in red light were impaired in the quadruple mutant for the 280
PHYTOCHROME INTERACTING FACTOR (PIF) genes PIF1 PIF3 PIF4 281
and PIF5 (pifq) indicating that the light-dependent regulation in red light was 282
mediated by the phytochrome and PIF signaling pathways (Fig S5) (Leivar et 283
al 2008) 284
The CK experiments indicated that CK induces the expression of all six GATA 285
genes (Fig 3) With an about three-fold increase in transcript abundance this 286
induction was strongest for GNL but could be observed for each of the other 287
GATA genes after a one to four hour CK treatment (Fig 3) Thus CK 288
promotes the expression of all six LLM-domain B-GATAs 289
290
Mutants of LLM-domain B-GATAs are impaired in hypocotyl elongation 291
Due to the strong regulation particularly of GNL but also of GNC in far-red red 292
and blue light conditions we examined the contribution of the GATA genes to 293
hypocotyl elongation by analyzing mutant seedlings in the different light 294
conditions in two different light intensities (Fig 4) Here we found that 295
quintuple mutant seedlings had a shorter hypocotyl than wild type seedlings in 296
white light as well as in red far-red and blue light (Fig 4) Notably these 297
phenotypes were not apparent or at least not significant in the gnc gnl 298
double mutant or the triple mutant suggesting that the respective other GATA 299
genes may contribute to the hypocotyl elongation phenotype in these 300
backgrounds (Fig 4) At the same time hypocotyl length of dark-grown 301
mutant seedlings was indistinguishable in all genotypes from that observed in 302
the wild type (Fig 4E and G) The short hypocotyl phenotype of the GATA 303
gene mutants was also interesting because we previously observed that the 304
LLM-domain B-GATA overexpressors had a longer hypocotyl than the wild 305
type when grown in white light (Behringer et al 2014) When analyzed in the 306
different light conditions we found the long hypocotyl phenotype of the GNL 307
overexpression line (GNLox) was also present in far-red and blue light but 308
interestingly not in red light where hypocotyls were even shorter than in the 309
wild type or the mutants when grown in weak red light conditions (Fig 4) 310
311
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
11
GATA gene mutants have differential gene expression defects The 312
strong induction of GNL gene expression by CK was already repeatedly 313
described and is the reason for the genersquos original designation CGA1 314
(CYTOKININ-INDUCED GATA1) (Naito et al 2007 Kollmer et al 2011) In 315
order to examine the role of the GATA genes in CK-responsive gene 316
expression we performed microarray experiments with 14 day-old wild type 317
plants gnc gnl and triple mutants as well as quintuple mutants treated for 60 318
min with the CK 6-BA (6-benzylaminopurine) or a corresponding mock solvent 319
control (Fig 5 and Fig 6 Supplemental Tables S1 and S2) We then 320
analyzed the resulting data with regard to differences in basal gene 321
expression (Fig 5) and with regard to gene expression differences after the 322
CK treatment in comparison to the mock control (Fig 6) For technical 323
reasons the experiment was conducted in two rounds the first experiment 324
included the Col-0 (1) wild type control the gnc gnl double mutant and the 325
quintuple mutant the second experiment included an independent Col-0 (2) 326
wild type control and the triple mutant When we examined the expression of 327
the CK-induced ARRs (A-TYPE RESPONSE REGULATORS) of the CK 328
signaling pathway we could confirm that the respective CK induction 329
experiments were comparably efficient between the two experiments and in 330
all genotypes tested (Fig S6A) We also examined the microarray dataset 331
with regard to the expression of a previously defined so-called ldquogolden listrdquo of 332
CK-regulated genes (Bhargava et al 2013) Over 70 of the 331 entities 333
derived from this list were significantly CK-regulated in our experiments 334
(Supplemental Table S3) When applying a two-fold expression change as 335
additional filter criterion 71 of these 331 entities were identified as up- (Col-0 336
1 and Col-0 2) and 39 (Col-0 1) and 25 entities (Col-0 2) as down-regulated 337
after CK treatment (Fig S6B and Supplemental Table S3) We thus 338
concluded that the CK treatments in both experiments were similarly efficient 339
and that the data sets could be used for comparative analyses 340
The comparison of the basal gene expression profiles of the wild type 341
samples and the mutants indicated that the (untreated) quintuple mutant 342
seedlings had by far the largest number of differentially regulated genes with 343
4722 down-regulated and 976 up-regulated entities when compared to the 344
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
12
Col-0 (1) wild type (Fig 5 and Supplemental Table S1) Furthermore the gnc 345
gnl double mutant contained 2458 down- and 294 up-regulated entities 346
respectively (Fig 5 and Supplemental Table S1) In comparison the number 347
of differentially expressed genes was comparatively minor in the triple mutant 348
which had only 323 down- and 278 up-regulated entities in this particular 349
experiment and selected physiological growth conditions (Fig 5) 350
The comparatively strong gene expression differences in the gnc gnl double 351
and the quintuple mutants and the fact that both mutants shared the gnc gnl 352
loss-of-function at the genetic level was reflected by the large overlap 353
between the differentially expressed gene sets of the two genotypes 354
Specifically 1922 of the 2458 down-regulated entities from gnc gnl were also 355
down-regulated in the quintuple mutant (Fig 5B) and 98 of the 294 entities 356
up-regulated in gnc gnl were also up-regulated in the quintuple mutant (Fig 357
5B) Conversely the overlap in gene expression defects between the triple 358
mutant and the quintuple mutant was higher than that between the triple 359
mutant and gnc gnl (Fig 5B) We therefore concluded that the gene 360
expression differences between the different GATA gene mutants was 361
strongest in the quintuple mutant and that the strong increase in the number 362
of differentially regulated genes exceeded by far the differences seen in the 363
gnc gnl double mutant and the triple mutant This supported our conclusion 364
from the phenotypic analyses that the GATA genes act in a functionally 365
redundant manner 366
Since we had observed increased gene expression of all six GATA genes 367
after CK-treatment we expected that CK-induced gene expression may be 368
compromised in the gata mutants (Fig 6) Indeed of the 996 entities that 369
were down-regulated after CK-treatment in the wild type (Col-0 1) only 173 370
(18 ) and 89 (9 ) remained CK-regulated in the quintuple and gnc gnl 371
mutant respectively (Fig 6A and Supplemental Table S2) At the same time 372
of the 188 entities that were down-regulated after CK treatment in the wild 373
type sample of the second experiment (Col-0 2) 83 (44 ) remained CK-374
regulated in the gata triple mutant (Fig 6B) The differential effect on gene 375
expression between the different mutants was less pronounced when 376
examining the CK-induced genes Here 49 (quintuple mutant) 59 (gnc 377
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
13
gnl) and 63 (triple mutant) of the CK-induced entities from the respective 378
wild types remained CK-regulated in the mutants (Fig 6A and B) In summary 379
we concluded that CK-regulated gene expression is impaired in the GATA 380
gene mutants 381
382
GATA gene mutants are defective in CK-regulated developmental 383
processes Based on the strong defects of gata mutants in CK-regulated 384
gene expression we searched for phenotypes that could be explained by 385
defects in CK response CK signaling was described as a regulator of 386
phyllotactic patterning in the shoot apical meristem of Arabidopsis (Besnard et 387
al 2014) In line with a role of the LLM-domain B-GATA genes in this process 388
we observed frequent aberrations from normal phyllotactic patterning in flower 389
positioning in triple and quintuple mutant inflorescences (Fig 7) Quantitative 390
analyses revealed aberrations from the canonical angle of ~1375deg in about 391
~30 of the petioles in triple and quintuple inflorescences (Fig 7D) At the 392
same time this patterning defect was not apparent in the gnc gnl double 393
mutant Since we also did not observe a quantitative increase in the severity 394
of this phenotype between the triple and the quintuple mutant we concluded 395
that GATA16 GATA17 and GATA17L may be responsible for this phenotype 396
(Fig 7D) 397
Leaf senescence is another CK-regulated process (Hwang et al 2012) In the 398
wild type CK levels are reduced during senescence and positive or negative 399
interference with the decrease in CK levels can promote and delay 400
senescence respectively (Hwang et al 2012) Senescence can be induced 401
by shifting light-grown plants to the dark and we had previously already shown 402
that leaf senescence is delayed in overexpression lines of LLM-domain B-403
GATAs (Behringer et al 2014) To examine possible senescence defects in 404
the gata mutants we floated leaves of wild type and mutant plants in the dark 405
on a mock solution or on a solution containing 005 microM or 01 microM 6-BA (Fig 406
8A ndash F) (Weaver and Amasino 2001) In line with the expectation that the 407
presence of CK in the medium would suppress the senescence process we 408
observed decreased senescence in CK-treated wild type leaves which we 409
quantitatively assessed by measuring chlorophyll levels in the mock- and CK-410
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
14
treated leaves (Fig 8F) Here we found that the effects of the senescence 411
treatment were less efficiently suppressed by CK treatments in the gnc gnl 412
double and in the quintuple mutant than in the wild type or the triple mutant 413
(Fig 8A - F) Similarly we observed a stronger effect on the decreases in 414
chlorophyll and total protein levels in the gnc gnl as well as the quintuple 415
mutant when we transferred light-grown plants for four days to the dark (Fig 416
8G ndash I) As previously reported GNL overexpressing plants (GNLox) had 417
senescence phenotypes that were antagonistic to the ones observed with the 418
mutants (Fig 8) (Behringer et al 2014) In the case of the leaf floating assay 419
CK-treatment had a stronger effect on greening and chlorophyll abundance in 420
GNLox than in the wild type (Fig 8A ndash F) When we transferred plants from 421
the light to the dark GNLox seedlings remained visibly green and did not 422
display the strong decrease in total protein abundance that we observed in 423
the wild type or the GATA gene mutants (Fig 8G ndash I) We thus concluded that 424
GATA gene mutants and overexpressors have antagonistic effects with 425
regard to the regulation of senescence and that GNC and GNL contribute 426
particularly strongly to this phenotype 427
CK induces greening in light-grown seedlings (Kobayashi et al 2012) To 428
examine to what extent CK can induce chlorophyll accumulation in the gata 429
mutants we grew plants for two weeks in constant light on medium containing 430
CK (5 nM BA) or a corresponding CK-free medium As expected chlorophyll 431
content was increased in wild type plants but this increase was neither 432
significant in the gnc gnl nor the triple or the quintuple mutants (Fig S7) We 433
thus concluded that the GATA factors are required for CK-induced greening in 434
Arabidopsis seedlings 435
It was previously reported that hypocotyl elongation could be repressed by 436
incubating dark-grown seedlings in the presence of CK (Su and Howell 1995) 437
Since the GATA gene mutants had displayed a hypocotyl elongation defect in 438
light-grown seedlings (Fig 4) we also explored the link between hypocotyl 439
elongation and CK However when we tested the effects of CK on hypocotyl 440
elongation in dark-grown seedlings we did not detect any significant 441
differences in the response between the different genotypes tested (Fig S8) 442
It is thus unlikely that the GATA genes participate in this elongation response 443
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
15
Branching is another phenotype that has been attributed to defects in CK 444
biosynthesis and response (El-Showk et al 2013) When we analyzed the 445
general habitus of the quintuple mutants we noted that they had an increased 446
number of secondary branches that was combined with a reduced plant 447
height (Fig 9A - C) Occasionally we also observed the outgrowth of 448
inflorescences in the axils of lateral inflorescences in the mutants whereas we 449
had not noted this phenotype in the wild type (Fig 9D and E) A quantitative 450
analysis of these phenotypes over all gata mutant phenotypes revealed that 451
the number of first and second order lateral branches increased with 452
increasing mutant complexity (Fig 9B) Here we observed strong effects 453
already in the gata17 gata17l mutant as well as the gnc gnl double mutant but 454
strongest defects in the triple and the quintuple mutant thus all five GATA 455
genes may contribute to this phenotype (Fig 9B) At the same time plant 456
height was reduced to a significant but still minor level in these mutants 457
suggesting that decreases in plant height which may have indirect 458
consequences on the general plant habitus and branching are not the 459
primary cause for altered branching patterns in the gata mutants (Fig 9C) It 460
is further important to note that the higher order GATA gene mutants initiated 461
fewer vegetative buds but had a higher number of growing buds as reflected 462
by the increased branch numbers (Fig 9F) Thus axillary bud formation is 463
promoted and bud outgrowth is repressed by the GATAs in the wild type 464
Another phenotype that became apparent during our analysis of the gata 465
mutant plant habitus was an increase in the angle formed between the 466
primary inflorescence and the lateral inflorescences (Fig 9G and H Fig S9) 467
gata mutants had a decreased angle formed between the primary 468
inflorescence and lateral inflorescences and this phenotype was strongest in 469
the quintuple mutant and intermediate in the gnc gnl and triple mutants (Fig 470
9G and H Fig S9) Interestingly we had previously observed increased 471
branch angles in GATA overexpression lines such as GNLox (Fig S9) 472
(Behringer et al 2014) We thus concluded that GATA gene mutants and 473
overexpressors antagonistically regulate branch angles in Arabidopsis 474
475
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
11
GATA gene mutants have differential gene expression defects The 312
strong induction of GNL gene expression by CK was already repeatedly 313
described and is the reason for the genersquos original designation CGA1 314
(CYTOKININ-INDUCED GATA1) (Naito et al 2007 Kollmer et al 2011) In 315
order to examine the role of the GATA genes in CK-responsive gene 316
expression we performed microarray experiments with 14 day-old wild type 317
plants gnc gnl and triple mutants as well as quintuple mutants treated for 60 318
min with the CK 6-BA (6-benzylaminopurine) or a corresponding mock solvent 319
control (Fig 5 and Fig 6 Supplemental Tables S1 and S2) We then 320
analyzed the resulting data with regard to differences in basal gene 321
expression (Fig 5) and with regard to gene expression differences after the 322
CK treatment in comparison to the mock control (Fig 6) For technical 323
reasons the experiment was conducted in two rounds the first experiment 324
included the Col-0 (1) wild type control the gnc gnl double mutant and the 325
quintuple mutant the second experiment included an independent Col-0 (2) 326
wild type control and the triple mutant When we examined the expression of 327
the CK-induced ARRs (A-TYPE RESPONSE REGULATORS) of the CK 328
signaling pathway we could confirm that the respective CK induction 329
experiments were comparably efficient between the two experiments and in 330
all genotypes tested (Fig S6A) We also examined the microarray dataset 331
with regard to the expression of a previously defined so-called ldquogolden listrdquo of 332
CK-regulated genes (Bhargava et al 2013) Over 70 of the 331 entities 333
derived from this list were significantly CK-regulated in our experiments 334
(Supplemental Table S3) When applying a two-fold expression change as 335
additional filter criterion 71 of these 331 entities were identified as up- (Col-0 336
1 and Col-0 2) and 39 (Col-0 1) and 25 entities (Col-0 2) as down-regulated 337
after CK treatment (Fig S6B and Supplemental Table S3) We thus 338
concluded that the CK treatments in both experiments were similarly efficient 339
and that the data sets could be used for comparative analyses 340
The comparison of the basal gene expression profiles of the wild type 341
samples and the mutants indicated that the (untreated) quintuple mutant 342
seedlings had by far the largest number of differentially regulated genes with 343
4722 down-regulated and 976 up-regulated entities when compared to the 344
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
12
Col-0 (1) wild type (Fig 5 and Supplemental Table S1) Furthermore the gnc 345
gnl double mutant contained 2458 down- and 294 up-regulated entities 346
respectively (Fig 5 and Supplemental Table S1) In comparison the number 347
of differentially expressed genes was comparatively minor in the triple mutant 348
which had only 323 down- and 278 up-regulated entities in this particular 349
experiment and selected physiological growth conditions (Fig 5) 350
The comparatively strong gene expression differences in the gnc gnl double 351
and the quintuple mutants and the fact that both mutants shared the gnc gnl 352
loss-of-function at the genetic level was reflected by the large overlap 353
between the differentially expressed gene sets of the two genotypes 354
Specifically 1922 of the 2458 down-regulated entities from gnc gnl were also 355
down-regulated in the quintuple mutant (Fig 5B) and 98 of the 294 entities 356
up-regulated in gnc gnl were also up-regulated in the quintuple mutant (Fig 357
5B) Conversely the overlap in gene expression defects between the triple 358
mutant and the quintuple mutant was higher than that between the triple 359
mutant and gnc gnl (Fig 5B) We therefore concluded that the gene 360
expression differences between the different GATA gene mutants was 361
strongest in the quintuple mutant and that the strong increase in the number 362
of differentially regulated genes exceeded by far the differences seen in the 363
gnc gnl double mutant and the triple mutant This supported our conclusion 364
from the phenotypic analyses that the GATA genes act in a functionally 365
redundant manner 366
Since we had observed increased gene expression of all six GATA genes 367
after CK-treatment we expected that CK-induced gene expression may be 368
compromised in the gata mutants (Fig 6) Indeed of the 996 entities that 369
were down-regulated after CK-treatment in the wild type (Col-0 1) only 173 370
(18 ) and 89 (9 ) remained CK-regulated in the quintuple and gnc gnl 371
mutant respectively (Fig 6A and Supplemental Table S2) At the same time 372
of the 188 entities that were down-regulated after CK treatment in the wild 373
type sample of the second experiment (Col-0 2) 83 (44 ) remained CK-374
regulated in the gata triple mutant (Fig 6B) The differential effect on gene 375
expression between the different mutants was less pronounced when 376
examining the CK-induced genes Here 49 (quintuple mutant) 59 (gnc 377
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
13
gnl) and 63 (triple mutant) of the CK-induced entities from the respective 378
wild types remained CK-regulated in the mutants (Fig 6A and B) In summary 379
we concluded that CK-regulated gene expression is impaired in the GATA 380
gene mutants 381
382
GATA gene mutants are defective in CK-regulated developmental 383
processes Based on the strong defects of gata mutants in CK-regulated 384
gene expression we searched for phenotypes that could be explained by 385
defects in CK response CK signaling was described as a regulator of 386
phyllotactic patterning in the shoot apical meristem of Arabidopsis (Besnard et 387
al 2014) In line with a role of the LLM-domain B-GATA genes in this process 388
we observed frequent aberrations from normal phyllotactic patterning in flower 389
positioning in triple and quintuple mutant inflorescences (Fig 7) Quantitative 390
analyses revealed aberrations from the canonical angle of ~1375deg in about 391
~30 of the petioles in triple and quintuple inflorescences (Fig 7D) At the 392
same time this patterning defect was not apparent in the gnc gnl double 393
mutant Since we also did not observe a quantitative increase in the severity 394
of this phenotype between the triple and the quintuple mutant we concluded 395
that GATA16 GATA17 and GATA17L may be responsible for this phenotype 396
(Fig 7D) 397
Leaf senescence is another CK-regulated process (Hwang et al 2012) In the 398
wild type CK levels are reduced during senescence and positive or negative 399
interference with the decrease in CK levels can promote and delay 400
senescence respectively (Hwang et al 2012) Senescence can be induced 401
by shifting light-grown plants to the dark and we had previously already shown 402
that leaf senescence is delayed in overexpression lines of LLM-domain B-403
GATAs (Behringer et al 2014) To examine possible senescence defects in 404
the gata mutants we floated leaves of wild type and mutant plants in the dark 405
on a mock solution or on a solution containing 005 microM or 01 microM 6-BA (Fig 406
8A ndash F) (Weaver and Amasino 2001) In line with the expectation that the 407
presence of CK in the medium would suppress the senescence process we 408
observed decreased senescence in CK-treated wild type leaves which we 409
quantitatively assessed by measuring chlorophyll levels in the mock- and CK-410
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
14
treated leaves (Fig 8F) Here we found that the effects of the senescence 411
treatment were less efficiently suppressed by CK treatments in the gnc gnl 412
double and in the quintuple mutant than in the wild type or the triple mutant 413
(Fig 8A - F) Similarly we observed a stronger effect on the decreases in 414
chlorophyll and total protein levels in the gnc gnl as well as the quintuple 415
mutant when we transferred light-grown plants for four days to the dark (Fig 416
8G ndash I) As previously reported GNL overexpressing plants (GNLox) had 417
senescence phenotypes that were antagonistic to the ones observed with the 418
mutants (Fig 8) (Behringer et al 2014) In the case of the leaf floating assay 419
CK-treatment had a stronger effect on greening and chlorophyll abundance in 420
GNLox than in the wild type (Fig 8A ndash F) When we transferred plants from 421
the light to the dark GNLox seedlings remained visibly green and did not 422
display the strong decrease in total protein abundance that we observed in 423
the wild type or the GATA gene mutants (Fig 8G ndash I) We thus concluded that 424
GATA gene mutants and overexpressors have antagonistic effects with 425
regard to the regulation of senescence and that GNC and GNL contribute 426
particularly strongly to this phenotype 427
CK induces greening in light-grown seedlings (Kobayashi et al 2012) To 428
examine to what extent CK can induce chlorophyll accumulation in the gata 429
mutants we grew plants for two weeks in constant light on medium containing 430
CK (5 nM BA) or a corresponding CK-free medium As expected chlorophyll 431
content was increased in wild type plants but this increase was neither 432
significant in the gnc gnl nor the triple or the quintuple mutants (Fig S7) We 433
thus concluded that the GATA factors are required for CK-induced greening in 434
Arabidopsis seedlings 435
It was previously reported that hypocotyl elongation could be repressed by 436
incubating dark-grown seedlings in the presence of CK (Su and Howell 1995) 437
Since the GATA gene mutants had displayed a hypocotyl elongation defect in 438
light-grown seedlings (Fig 4) we also explored the link between hypocotyl 439
elongation and CK However when we tested the effects of CK on hypocotyl 440
elongation in dark-grown seedlings we did not detect any significant 441
differences in the response between the different genotypes tested (Fig S8) 442
It is thus unlikely that the GATA genes participate in this elongation response 443
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
15
Branching is another phenotype that has been attributed to defects in CK 444
biosynthesis and response (El-Showk et al 2013) When we analyzed the 445
general habitus of the quintuple mutants we noted that they had an increased 446
number of secondary branches that was combined with a reduced plant 447
height (Fig 9A - C) Occasionally we also observed the outgrowth of 448
inflorescences in the axils of lateral inflorescences in the mutants whereas we 449
had not noted this phenotype in the wild type (Fig 9D and E) A quantitative 450
analysis of these phenotypes over all gata mutant phenotypes revealed that 451
the number of first and second order lateral branches increased with 452
increasing mutant complexity (Fig 9B) Here we observed strong effects 453
already in the gata17 gata17l mutant as well as the gnc gnl double mutant but 454
strongest defects in the triple and the quintuple mutant thus all five GATA 455
genes may contribute to this phenotype (Fig 9B) At the same time plant 456
height was reduced to a significant but still minor level in these mutants 457
suggesting that decreases in plant height which may have indirect 458
consequences on the general plant habitus and branching are not the 459
primary cause for altered branching patterns in the gata mutants (Fig 9C) It 460
is further important to note that the higher order GATA gene mutants initiated 461
fewer vegetative buds but had a higher number of growing buds as reflected 462
by the increased branch numbers (Fig 9F) Thus axillary bud formation is 463
promoted and bud outgrowth is repressed by the GATAs in the wild type 464
Another phenotype that became apparent during our analysis of the gata 465
mutant plant habitus was an increase in the angle formed between the 466
primary inflorescence and the lateral inflorescences (Fig 9G and H Fig S9) 467
gata mutants had a decreased angle formed between the primary 468
inflorescence and lateral inflorescences and this phenotype was strongest in 469
the quintuple mutant and intermediate in the gnc gnl and triple mutants (Fig 470
9G and H Fig S9) Interestingly we had previously observed increased 471
branch angles in GATA overexpression lines such as GNLox (Fig S9) 472
(Behringer et al 2014) We thus concluded that GATA gene mutants and 473
overexpressors antagonistically regulate branch angles in Arabidopsis 474
475
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
12
Col-0 (1) wild type (Fig 5 and Supplemental Table S1) Furthermore the gnc 345
gnl double mutant contained 2458 down- and 294 up-regulated entities 346
respectively (Fig 5 and Supplemental Table S1) In comparison the number 347
of differentially expressed genes was comparatively minor in the triple mutant 348
which had only 323 down- and 278 up-regulated entities in this particular 349
experiment and selected physiological growth conditions (Fig 5) 350
The comparatively strong gene expression differences in the gnc gnl double 351
and the quintuple mutants and the fact that both mutants shared the gnc gnl 352
loss-of-function at the genetic level was reflected by the large overlap 353
between the differentially expressed gene sets of the two genotypes 354
Specifically 1922 of the 2458 down-regulated entities from gnc gnl were also 355
down-regulated in the quintuple mutant (Fig 5B) and 98 of the 294 entities 356
up-regulated in gnc gnl were also up-regulated in the quintuple mutant (Fig 357
5B) Conversely the overlap in gene expression defects between the triple 358
mutant and the quintuple mutant was higher than that between the triple 359
mutant and gnc gnl (Fig 5B) We therefore concluded that the gene 360
expression differences between the different GATA gene mutants was 361
strongest in the quintuple mutant and that the strong increase in the number 362
of differentially regulated genes exceeded by far the differences seen in the 363
gnc gnl double mutant and the triple mutant This supported our conclusion 364
from the phenotypic analyses that the GATA genes act in a functionally 365
redundant manner 366
Since we had observed increased gene expression of all six GATA genes 367
after CK-treatment we expected that CK-induced gene expression may be 368
compromised in the gata mutants (Fig 6) Indeed of the 996 entities that 369
were down-regulated after CK-treatment in the wild type (Col-0 1) only 173 370
(18 ) and 89 (9 ) remained CK-regulated in the quintuple and gnc gnl 371
mutant respectively (Fig 6A and Supplemental Table S2) At the same time 372
of the 188 entities that were down-regulated after CK treatment in the wild 373
type sample of the second experiment (Col-0 2) 83 (44 ) remained CK-374
regulated in the gata triple mutant (Fig 6B) The differential effect on gene 375
expression between the different mutants was less pronounced when 376
examining the CK-induced genes Here 49 (quintuple mutant) 59 (gnc 377
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
13
gnl) and 63 (triple mutant) of the CK-induced entities from the respective 378
wild types remained CK-regulated in the mutants (Fig 6A and B) In summary 379
we concluded that CK-regulated gene expression is impaired in the GATA 380
gene mutants 381
382
GATA gene mutants are defective in CK-regulated developmental 383
processes Based on the strong defects of gata mutants in CK-regulated 384
gene expression we searched for phenotypes that could be explained by 385
defects in CK response CK signaling was described as a regulator of 386
phyllotactic patterning in the shoot apical meristem of Arabidopsis (Besnard et 387
al 2014) In line with a role of the LLM-domain B-GATA genes in this process 388
we observed frequent aberrations from normal phyllotactic patterning in flower 389
positioning in triple and quintuple mutant inflorescences (Fig 7) Quantitative 390
analyses revealed aberrations from the canonical angle of ~1375deg in about 391
~30 of the petioles in triple and quintuple inflorescences (Fig 7D) At the 392
same time this patterning defect was not apparent in the gnc gnl double 393
mutant Since we also did not observe a quantitative increase in the severity 394
of this phenotype between the triple and the quintuple mutant we concluded 395
that GATA16 GATA17 and GATA17L may be responsible for this phenotype 396
(Fig 7D) 397
Leaf senescence is another CK-regulated process (Hwang et al 2012) In the 398
wild type CK levels are reduced during senescence and positive or negative 399
interference with the decrease in CK levels can promote and delay 400
senescence respectively (Hwang et al 2012) Senescence can be induced 401
by shifting light-grown plants to the dark and we had previously already shown 402
that leaf senescence is delayed in overexpression lines of LLM-domain B-403
GATAs (Behringer et al 2014) To examine possible senescence defects in 404
the gata mutants we floated leaves of wild type and mutant plants in the dark 405
on a mock solution or on a solution containing 005 microM or 01 microM 6-BA (Fig 406
8A ndash F) (Weaver and Amasino 2001) In line with the expectation that the 407
presence of CK in the medium would suppress the senescence process we 408
observed decreased senescence in CK-treated wild type leaves which we 409
quantitatively assessed by measuring chlorophyll levels in the mock- and CK-410
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
14
treated leaves (Fig 8F) Here we found that the effects of the senescence 411
treatment were less efficiently suppressed by CK treatments in the gnc gnl 412
double and in the quintuple mutant than in the wild type or the triple mutant 413
(Fig 8A - F) Similarly we observed a stronger effect on the decreases in 414
chlorophyll and total protein levels in the gnc gnl as well as the quintuple 415
mutant when we transferred light-grown plants for four days to the dark (Fig 416
8G ndash I) As previously reported GNL overexpressing plants (GNLox) had 417
senescence phenotypes that were antagonistic to the ones observed with the 418
mutants (Fig 8) (Behringer et al 2014) In the case of the leaf floating assay 419
CK-treatment had a stronger effect on greening and chlorophyll abundance in 420
GNLox than in the wild type (Fig 8A ndash F) When we transferred plants from 421
the light to the dark GNLox seedlings remained visibly green and did not 422
display the strong decrease in total protein abundance that we observed in 423
the wild type or the GATA gene mutants (Fig 8G ndash I) We thus concluded that 424
GATA gene mutants and overexpressors have antagonistic effects with 425
regard to the regulation of senescence and that GNC and GNL contribute 426
particularly strongly to this phenotype 427
CK induces greening in light-grown seedlings (Kobayashi et al 2012) To 428
examine to what extent CK can induce chlorophyll accumulation in the gata 429
mutants we grew plants for two weeks in constant light on medium containing 430
CK (5 nM BA) or a corresponding CK-free medium As expected chlorophyll 431
content was increased in wild type plants but this increase was neither 432
significant in the gnc gnl nor the triple or the quintuple mutants (Fig S7) We 433
thus concluded that the GATA factors are required for CK-induced greening in 434
Arabidopsis seedlings 435
It was previously reported that hypocotyl elongation could be repressed by 436
incubating dark-grown seedlings in the presence of CK (Su and Howell 1995) 437
Since the GATA gene mutants had displayed a hypocotyl elongation defect in 438
light-grown seedlings (Fig 4) we also explored the link between hypocotyl 439
elongation and CK However when we tested the effects of CK on hypocotyl 440
elongation in dark-grown seedlings we did not detect any significant 441
differences in the response between the different genotypes tested (Fig S8) 442
It is thus unlikely that the GATA genes participate in this elongation response 443
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
15
Branching is another phenotype that has been attributed to defects in CK 444
biosynthesis and response (El-Showk et al 2013) When we analyzed the 445
general habitus of the quintuple mutants we noted that they had an increased 446
number of secondary branches that was combined with a reduced plant 447
height (Fig 9A - C) Occasionally we also observed the outgrowth of 448
inflorescences in the axils of lateral inflorescences in the mutants whereas we 449
had not noted this phenotype in the wild type (Fig 9D and E) A quantitative 450
analysis of these phenotypes over all gata mutant phenotypes revealed that 451
the number of first and second order lateral branches increased with 452
increasing mutant complexity (Fig 9B) Here we observed strong effects 453
already in the gata17 gata17l mutant as well as the gnc gnl double mutant but 454
strongest defects in the triple and the quintuple mutant thus all five GATA 455
genes may contribute to this phenotype (Fig 9B) At the same time plant 456
height was reduced to a significant but still minor level in these mutants 457
suggesting that decreases in plant height which may have indirect 458
consequences on the general plant habitus and branching are not the 459
primary cause for altered branching patterns in the gata mutants (Fig 9C) It 460
is further important to note that the higher order GATA gene mutants initiated 461
fewer vegetative buds but had a higher number of growing buds as reflected 462
by the increased branch numbers (Fig 9F) Thus axillary bud formation is 463
promoted and bud outgrowth is repressed by the GATAs in the wild type 464
Another phenotype that became apparent during our analysis of the gata 465
mutant plant habitus was an increase in the angle formed between the 466
primary inflorescence and the lateral inflorescences (Fig 9G and H Fig S9) 467
gata mutants had a decreased angle formed between the primary 468
inflorescence and lateral inflorescences and this phenotype was strongest in 469
the quintuple mutant and intermediate in the gnc gnl and triple mutants (Fig 470
9G and H Fig S9) Interestingly we had previously observed increased 471
branch angles in GATA overexpression lines such as GNLox (Fig S9) 472
(Behringer et al 2014) We thus concluded that GATA gene mutants and 473
overexpressors antagonistically regulate branch angles in Arabidopsis 474
475
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
13
gnl) and 63 (triple mutant) of the CK-induced entities from the respective 378
wild types remained CK-regulated in the mutants (Fig 6A and B) In summary 379
we concluded that CK-regulated gene expression is impaired in the GATA 380
gene mutants 381
382
GATA gene mutants are defective in CK-regulated developmental 383
processes Based on the strong defects of gata mutants in CK-regulated 384
gene expression we searched for phenotypes that could be explained by 385
defects in CK response CK signaling was described as a regulator of 386
phyllotactic patterning in the shoot apical meristem of Arabidopsis (Besnard et 387
al 2014) In line with a role of the LLM-domain B-GATA genes in this process 388
we observed frequent aberrations from normal phyllotactic patterning in flower 389
positioning in triple and quintuple mutant inflorescences (Fig 7) Quantitative 390
analyses revealed aberrations from the canonical angle of ~1375deg in about 391
~30 of the petioles in triple and quintuple inflorescences (Fig 7D) At the 392
same time this patterning defect was not apparent in the gnc gnl double 393
mutant Since we also did not observe a quantitative increase in the severity 394
of this phenotype between the triple and the quintuple mutant we concluded 395
that GATA16 GATA17 and GATA17L may be responsible for this phenotype 396
(Fig 7D) 397
Leaf senescence is another CK-regulated process (Hwang et al 2012) In the 398
wild type CK levels are reduced during senescence and positive or negative 399
interference with the decrease in CK levels can promote and delay 400
senescence respectively (Hwang et al 2012) Senescence can be induced 401
by shifting light-grown plants to the dark and we had previously already shown 402
that leaf senescence is delayed in overexpression lines of LLM-domain B-403
GATAs (Behringer et al 2014) To examine possible senescence defects in 404
the gata mutants we floated leaves of wild type and mutant plants in the dark 405
on a mock solution or on a solution containing 005 microM or 01 microM 6-BA (Fig 406
8A ndash F) (Weaver and Amasino 2001) In line with the expectation that the 407
presence of CK in the medium would suppress the senescence process we 408
observed decreased senescence in CK-treated wild type leaves which we 409
quantitatively assessed by measuring chlorophyll levels in the mock- and CK-410
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
14
treated leaves (Fig 8F) Here we found that the effects of the senescence 411
treatment were less efficiently suppressed by CK treatments in the gnc gnl 412
double and in the quintuple mutant than in the wild type or the triple mutant 413
(Fig 8A - F) Similarly we observed a stronger effect on the decreases in 414
chlorophyll and total protein levels in the gnc gnl as well as the quintuple 415
mutant when we transferred light-grown plants for four days to the dark (Fig 416
8G ndash I) As previously reported GNL overexpressing plants (GNLox) had 417
senescence phenotypes that were antagonistic to the ones observed with the 418
mutants (Fig 8) (Behringer et al 2014) In the case of the leaf floating assay 419
CK-treatment had a stronger effect on greening and chlorophyll abundance in 420
GNLox than in the wild type (Fig 8A ndash F) When we transferred plants from 421
the light to the dark GNLox seedlings remained visibly green and did not 422
display the strong decrease in total protein abundance that we observed in 423
the wild type or the GATA gene mutants (Fig 8G ndash I) We thus concluded that 424
GATA gene mutants and overexpressors have antagonistic effects with 425
regard to the regulation of senescence and that GNC and GNL contribute 426
particularly strongly to this phenotype 427
CK induces greening in light-grown seedlings (Kobayashi et al 2012) To 428
examine to what extent CK can induce chlorophyll accumulation in the gata 429
mutants we grew plants for two weeks in constant light on medium containing 430
CK (5 nM BA) or a corresponding CK-free medium As expected chlorophyll 431
content was increased in wild type plants but this increase was neither 432
significant in the gnc gnl nor the triple or the quintuple mutants (Fig S7) We 433
thus concluded that the GATA factors are required for CK-induced greening in 434
Arabidopsis seedlings 435
It was previously reported that hypocotyl elongation could be repressed by 436
incubating dark-grown seedlings in the presence of CK (Su and Howell 1995) 437
Since the GATA gene mutants had displayed a hypocotyl elongation defect in 438
light-grown seedlings (Fig 4) we also explored the link between hypocotyl 439
elongation and CK However when we tested the effects of CK on hypocotyl 440
elongation in dark-grown seedlings we did not detect any significant 441
differences in the response between the different genotypes tested (Fig S8) 442
It is thus unlikely that the GATA genes participate in this elongation response 443
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
15
Branching is another phenotype that has been attributed to defects in CK 444
biosynthesis and response (El-Showk et al 2013) When we analyzed the 445
general habitus of the quintuple mutants we noted that they had an increased 446
number of secondary branches that was combined with a reduced plant 447
height (Fig 9A - C) Occasionally we also observed the outgrowth of 448
inflorescences in the axils of lateral inflorescences in the mutants whereas we 449
had not noted this phenotype in the wild type (Fig 9D and E) A quantitative 450
analysis of these phenotypes over all gata mutant phenotypes revealed that 451
the number of first and second order lateral branches increased with 452
increasing mutant complexity (Fig 9B) Here we observed strong effects 453
already in the gata17 gata17l mutant as well as the gnc gnl double mutant but 454
strongest defects in the triple and the quintuple mutant thus all five GATA 455
genes may contribute to this phenotype (Fig 9B) At the same time plant 456
height was reduced to a significant but still minor level in these mutants 457
suggesting that decreases in plant height which may have indirect 458
consequences on the general plant habitus and branching are not the 459
primary cause for altered branching patterns in the gata mutants (Fig 9C) It 460
is further important to note that the higher order GATA gene mutants initiated 461
fewer vegetative buds but had a higher number of growing buds as reflected 462
by the increased branch numbers (Fig 9F) Thus axillary bud formation is 463
promoted and bud outgrowth is repressed by the GATAs in the wild type 464
Another phenotype that became apparent during our analysis of the gata 465
mutant plant habitus was an increase in the angle formed between the 466
primary inflorescence and the lateral inflorescences (Fig 9G and H Fig S9) 467
gata mutants had a decreased angle formed between the primary 468
inflorescence and lateral inflorescences and this phenotype was strongest in 469
the quintuple mutant and intermediate in the gnc gnl and triple mutants (Fig 470
9G and H Fig S9) Interestingly we had previously observed increased 471
branch angles in GATA overexpression lines such as GNLox (Fig S9) 472
(Behringer et al 2014) We thus concluded that GATA gene mutants and 473
overexpressors antagonistically regulate branch angles in Arabidopsis 474
475
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
14
treated leaves (Fig 8F) Here we found that the effects of the senescence 411
treatment were less efficiently suppressed by CK treatments in the gnc gnl 412
double and in the quintuple mutant than in the wild type or the triple mutant 413
(Fig 8A - F) Similarly we observed a stronger effect on the decreases in 414
chlorophyll and total protein levels in the gnc gnl as well as the quintuple 415
mutant when we transferred light-grown plants for four days to the dark (Fig 416
8G ndash I) As previously reported GNL overexpressing plants (GNLox) had 417
senescence phenotypes that were antagonistic to the ones observed with the 418
mutants (Fig 8) (Behringer et al 2014) In the case of the leaf floating assay 419
CK-treatment had a stronger effect on greening and chlorophyll abundance in 420
GNLox than in the wild type (Fig 8A ndash F) When we transferred plants from 421
the light to the dark GNLox seedlings remained visibly green and did not 422
display the strong decrease in total protein abundance that we observed in 423
the wild type or the GATA gene mutants (Fig 8G ndash I) We thus concluded that 424
GATA gene mutants and overexpressors have antagonistic effects with 425
regard to the regulation of senescence and that GNC and GNL contribute 426
particularly strongly to this phenotype 427
CK induces greening in light-grown seedlings (Kobayashi et al 2012) To 428
examine to what extent CK can induce chlorophyll accumulation in the gata 429
mutants we grew plants for two weeks in constant light on medium containing 430
CK (5 nM BA) or a corresponding CK-free medium As expected chlorophyll 431
content was increased in wild type plants but this increase was neither 432
significant in the gnc gnl nor the triple or the quintuple mutants (Fig S7) We 433
thus concluded that the GATA factors are required for CK-induced greening in 434
Arabidopsis seedlings 435
It was previously reported that hypocotyl elongation could be repressed by 436
incubating dark-grown seedlings in the presence of CK (Su and Howell 1995) 437
Since the GATA gene mutants had displayed a hypocotyl elongation defect in 438
light-grown seedlings (Fig 4) we also explored the link between hypocotyl 439
elongation and CK However when we tested the effects of CK on hypocotyl 440
elongation in dark-grown seedlings we did not detect any significant 441
differences in the response between the different genotypes tested (Fig S8) 442
It is thus unlikely that the GATA genes participate in this elongation response 443
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
15
Branching is another phenotype that has been attributed to defects in CK 444
biosynthesis and response (El-Showk et al 2013) When we analyzed the 445
general habitus of the quintuple mutants we noted that they had an increased 446
number of secondary branches that was combined with a reduced plant 447
height (Fig 9A - C) Occasionally we also observed the outgrowth of 448
inflorescences in the axils of lateral inflorescences in the mutants whereas we 449
had not noted this phenotype in the wild type (Fig 9D and E) A quantitative 450
analysis of these phenotypes over all gata mutant phenotypes revealed that 451
the number of first and second order lateral branches increased with 452
increasing mutant complexity (Fig 9B) Here we observed strong effects 453
already in the gata17 gata17l mutant as well as the gnc gnl double mutant but 454
strongest defects in the triple and the quintuple mutant thus all five GATA 455
genes may contribute to this phenotype (Fig 9B) At the same time plant 456
height was reduced to a significant but still minor level in these mutants 457
suggesting that decreases in plant height which may have indirect 458
consequences on the general plant habitus and branching are not the 459
primary cause for altered branching patterns in the gata mutants (Fig 9C) It 460
is further important to note that the higher order GATA gene mutants initiated 461
fewer vegetative buds but had a higher number of growing buds as reflected 462
by the increased branch numbers (Fig 9F) Thus axillary bud formation is 463
promoted and bud outgrowth is repressed by the GATAs in the wild type 464
Another phenotype that became apparent during our analysis of the gata 465
mutant plant habitus was an increase in the angle formed between the 466
primary inflorescence and the lateral inflorescences (Fig 9G and H Fig S9) 467
gata mutants had a decreased angle formed between the primary 468
inflorescence and lateral inflorescences and this phenotype was strongest in 469
the quintuple mutant and intermediate in the gnc gnl and triple mutants (Fig 470
9G and H Fig S9) Interestingly we had previously observed increased 471
branch angles in GATA overexpression lines such as GNLox (Fig S9) 472
(Behringer et al 2014) We thus concluded that GATA gene mutants and 473
overexpressors antagonistically regulate branch angles in Arabidopsis 474
475
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
15
Branching is another phenotype that has been attributed to defects in CK 444
biosynthesis and response (El-Showk et al 2013) When we analyzed the 445
general habitus of the quintuple mutants we noted that they had an increased 446
number of secondary branches that was combined with a reduced plant 447
height (Fig 9A - C) Occasionally we also observed the outgrowth of 448
inflorescences in the axils of lateral inflorescences in the mutants whereas we 449
had not noted this phenotype in the wild type (Fig 9D and E) A quantitative 450
analysis of these phenotypes over all gata mutant phenotypes revealed that 451
the number of first and second order lateral branches increased with 452
increasing mutant complexity (Fig 9B) Here we observed strong effects 453
already in the gata17 gata17l mutant as well as the gnc gnl double mutant but 454
strongest defects in the triple and the quintuple mutant thus all five GATA 455
genes may contribute to this phenotype (Fig 9B) At the same time plant 456
height was reduced to a significant but still minor level in these mutants 457
suggesting that decreases in plant height which may have indirect 458
consequences on the general plant habitus and branching are not the 459
primary cause for altered branching patterns in the gata mutants (Fig 9C) It 460
is further important to note that the higher order GATA gene mutants initiated 461
fewer vegetative buds but had a higher number of growing buds as reflected 462
by the increased branch numbers (Fig 9F) Thus axillary bud formation is 463
promoted and bud outgrowth is repressed by the GATAs in the wild type 464
Another phenotype that became apparent during our analysis of the gata 465
mutant plant habitus was an increase in the angle formed between the 466
primary inflorescence and the lateral inflorescences (Fig 9G and H Fig S9) 467
gata mutants had a decreased angle formed between the primary 468
inflorescence and lateral inflorescences and this phenotype was strongest in 469
the quintuple mutant and intermediate in the gnc gnl and triple mutants (Fig 470
9G and H Fig S9) Interestingly we had previously observed increased 471
branch angles in GATA overexpression lines such as GNLox (Fig S9) 472
(Behringer et al 2014) We thus concluded that GATA gene mutants and 473
overexpressors antagonistically regulate branch angles in Arabidopsis 474
475
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
16
GATA genes regulate petal and sepal numbers as well as silique length 476
CK signaling has recently been implicated in the control of floral development 477
(Han et al 2014) When we analyzed gata mutant flowers in more detail we 478
noted with interest aberrations in floral organ numbers in comparison to the 479
wild type (Fig 10A) Whereas wild type flowers have generally four sepals 480
and four petals and only rarely deviate from this pattern (in our study in 481
around 2 of the cases) a strong increase in flowers with more than four 482
(generally five in rare cases six) petals and sepals became apparent in the 483
higher order gata mutants (Fig 10A and Fig S10A) In the gata17 gata17l 484
mutants we observed flowers with more than four sepals and petals in 12 of 485
all flowers and this number was slightly enhanced in the triple mutant (13 486
petals) and the quintuple mutant (14 petals) (Fig 10A and Fig S10A) 487
Importantly the single mutants with the clearest apparent effect were the 488
gata17 and gata17l mutants and inversely mutations in GNC and GNL 489
contributed only to a minor extent to this phenotype (Fig S10A) In summary 490
this analysis revealed that GATA17 and GATA17L make a particularly strong 491
contribution to the floral morphology phenotype of the gata mutants 492
Since we had previously noted that overexpression lines of LLM-domain B-493
GATAs have short siliques and a reduced number of seeds per silique we 494
also analyzed silique length and seed set in the gata mutants (Fig S10B - D) 495
In line with an antagonistic regulation of these traits in the gata mutants we 496
measured significant increases in seed set in the siliques of the gnc gnl the 497
triple as well as the quintuple mutants and we detected significant increases 498
in silique length in all mutant backgrounds (Fig S10B - D) We thus concluded 499
that the GATA genes repress silique elongation and seed set in the wild type 500
at least to a minor extent A differential contribution of individual GATAs to 501
these phenotypes could however not be resolved here 502
503
GATA gene mutants have antagonistic effects on flowering time in long 504
and short days Accelerated flowering in plants grown under long-day 505
conditions was another phenotype previously described for gnc gnl mutants 506
(Richter et al 2010 Richter et al 2013) Conversely overexpression of the 507
LLM-domain B-GATAs causes a strong delay in flowering (Richter et al 508
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
17
2010 Richter et al 2013 Behringer et al 2014) To examine the contribution 509
of the other GATA factors to the flowering time phenotype we grew the gata 510
mutant series in long day and short day conditions with a 16 hr and 8 hr light 511
period respectively (Fig 10B and C Fig S11) Interestingly we observed a 512
gradual acceleration of flowering in long day-grown plants that was weakest in 513
the single mutants more pronounced in the double triple quadruple mutants 514
and strongest in the quintuple mutants (Fig 10B and C Figs S11 and S12) 515
We thus concluded that all LLM-domain B-GATA genes examined here may 516
contribute to the repression of flowering in long day-grown plants 517
Interestingly the effects of the reduced GATA gene function on flowering time 518
in short day conditions were antagonistic to those observed in long day-grown 519
plants in that the complex GATA gene mutants flowered much later than the 520
wild type or the less complex mutant combinations when flowering was 521
counted in days (Fig 10B and C Figs S11 and S12) We thus concluded that 522
the LLM-domain B-GATAs factors regulate flowering time in long and short 523
day conditions but in view of the antagonistic nature of this regulation that 524
their contribution to flowering time control may be complex 525
526
Cross-regulation of GATA gene expression Since several of our genetic 527
analyses had suggested a functional redundancy between the GATA genes 528
we looked for evidences of a cross-regulation of GATA gene expression in the 529
gnc gnl double mutant background There we detected indeed an increased 530
transcript abundance of GATA16 GATA17 and GATA17L in the gnc gnl 531
mutant which could be compensatory for the loss of GNC and GNL (Fig 11A) 532
At the same time GATA15 GATA16 and GATA17L gene expression levels 533
were significantly reduced in GNL overexpression lines (GNLox) suggesting 534
that GNL may regulate the expression of these genes 535
Since these observations indicated that molecular mechanisms may exist that 536
compensate for the loss of individual LLM-domain B-GATA genes eg in the 537
gnc gnl mutants we examined the binding of GNL to the LLM-domain GATA 538
gene promoters after ChIP-seq analysis (chromatin immunoprecipitation 539
followed by DNA sequencing) Here we found that at least three of the six 540
LLM-domain B-GATA genes were bound by GNL after immunoprecipitation of 541
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
18
HA-tagged GNL from transgenic plants expressing a functional GNLHA 542
transgene from a GNL promoter fragment in the gnc gnl background 543
(pGNLGNLHA gnc gnl Fig 11B) Through subsequent ChIP-PCR studies 544
targeting selected regions upstream from GNC GNL and GATA17 we could 545
confirm this binding through independent analyses (Fig 11C) Thus LLM-546
domain B-GATA factors may directly regulate their own expression as well as 547
that of other family members 548
549
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
19
DISCUSSION 550
In the present study we have characterized the six-membered family of LLM-551
domain B-GATAs from Arabidopsis Whereas the family has diverged when 552
compared between Arabidopsis tomato barley and Brachypodium a clear 553
conservation of this protein family can be observed within the Brassicaceae 554
family (Fig S1) (Behringer et al 2014) Within the Brassicaceae GNC and 555
GNL GATA15 and GATA16 as well as GATA17 and GATA17L form closely 556
related LLM-domain B-GATA pairs In a previous study we had comparatively 557
analyzed transgenic Arabidopsis lines expressing different members of the 558
LLM-domain B-GATA gene family from Arabidopsis barley and tomato and 559
concluded that these genes are functionally redundant at the biochemical 560
level since their overexpression resulted in highly similar phenotypes 561
(Behringer et al 2014) Several of the phenotypes analyzed here namely 562
greening (Fig 1D-F) hypocotyl elongation (Fig 4) plant height branching 563
patterns (Fig 9) branch angles (Fig 9 and Fig S9) silique length seed set 564
(Fig S10) flowering time (Fig 10) as well as senescence (Fig 8) are 565
strongest in the gata quintuple mutant and weaker or in some cases not even 566
apparent in the less complex gata mutant combinations Thus in line with this 567
previous study our present analysis shows that the LLM-domain B-GATA 568
genes from Arabidopsis have a shared biological function in the control of 569
multiple developmental pathways 570
Due to the high complexity of the available range of single double triple 571
quadruple and quintuple mutants we restricted many of our phenotypic 572
analyses to the gnc gnl double the complementary triple and the quintuple 573
mutants It can nevertheless be suggested based on the presently available 574
data that some phenotypes are predominantly controlled by a single LLM-575
domain B-GATA or rather an LLM-domain B-GATA gene pair Phyllotactic 576
patterning as assessed here in the inflorescence is impaired in the gata triple 577
but not in the gnc gnl double mutant (Fig 7) Similarly the increases in floral 578
organ numbers are already very prominent in the gata17 gata17l double 579
mutants and not much more enhanced in the gata triple or quintuple mutant 580
that includes the gata17 gata17l mutations (Fig 10A and Fig S10A) Thus 581
the LLM-domain B-GATA genes have shared overlapping but also distinct 582
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
20
roles in the control of plant development The fact that GATA15 gene function 583
is not impaired in the selected GATA15 gene insertion allele does not allow us 584
to draw any direct conclusions on the biological function of this gene based on 585
the data presented here Furthermore the fact that the mutant alleles of 586
GATA16 and GATA17 used in the complex mutants are only partially 587
defective in GATA gene expression could suggest that even stronger 588
phenotypes may be observed if loss-of-function alleles were used The two 589
strong alleles for GATA15 (gata15-2) and GATA17 (gata17-2) isolated here 590
but analyzed only as single mutants may help in the future to understand the 591
biological function of these GATA genes at further depth 592
It is interesting that the overexpression lines and the mutants of these GATA 593
genes frequently have antagonistic phenotypes This study in combination 594
with a previous study identifies such antagonistically regulated phenotypes in 595
the control of greening hypocotyl elongation branch angle formation 596
flowering time and senescence (Richter et al 2010 Behringer et al 2014) 597
Particularly in the case of transcription factors where the loss-of-function or 598
overexpression may affect the expression of many downstream targets genes 599
and developmental responses triggered by these target genes one may not 600
expect such a clear antagonistic relationship between mutants with reduced 601
gene functions and the overexpressors In view of the fact that 602
overexpressors likely also control the expression of genes that are normally 603
not targeted by this transcription factor when expressed from their native 604
promoter (off-targets) this observation is even more striking It may suggest 605
that the GATAs examined here control developmental responses in a dosage-606
dependent manner together with other critical factors In this context the 607
presence and abundance of the GATAs would just modulate the response but 608
would by itself not be sufficient to trigger it Further analyses will be required 609
to gain a more detailed understanding of the mode of action of the LLM-610
domain B-GATA factors in the control of gene expression 611
We had previously shown that the LLM-domain B-GATAs GNC and GNL 612
repress flowering since their loss-of-function mutants and overexpressors 613
displayed an early and late flowering phenotype respectively when plants 614
were grown in long-day conditions (Richter et al 2010 Richter et al 2013) 615
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
21
We furthermore showed that the control of flowering is partially mediated by 616
the flowering time regulator SOC1 (SUPPRESSOR OF OVEREXPRESSION 617
OF CONSTANS1) which acts downstream from GNC and GNL and whose 618
promoter can be directly bound by GNC and GNL (Richter et al 2013) In line 619
with these previous observations we show here that the complex GATA gene 620
mutants flower even earlier than the gnc gnl mutant further substantiating our 621
conclusion that LLM-domain B-GATAs are flowering time regulators in 622
Arabidopsis (Fig 10B and Fig S11) Surprisingly the complex gata mutants 623
flowered late when grown in short day conditions and their phenotype was 624
thereby antagonistic to the phenotype observed in long days (Fig 10C and 625
Fig S9) The reason for this antagonistic flowering time behavior remains to 626
be resolved but a complex scenario may be envisioned We also do not want 627
to rule out that physiological parameters become limiting in the induction of 628
flowering in the gata mutants Long term defects as a result of their reduced 629
greening may play a limiting role during the extended growth in short days 630
with short illumination periods that are not limiting when plants are grown in 631
long days 632
Several of our phenotypic analyses suggested that the LLM-domain B-GATAs 633
act in concert to control specific phenotypes The fact that at least some of the 634
phenotypes were most pronounced in the quintuple mutant and not apparent 635
or only weak in the less complex mutants suggests that the LLM-domain B-636
GATAs may cross-regulate their expression and that presence and absence 637
of one GATA could interfere with the abundance of other GATA family 638
members We have analyzed this hypothesis here by examining the 639
expression of the LLM-domain B-GATAs GATA15 GATA16 GATA17 and 640
GATA17L in the gnc gnl mutant and in the GNL overexpression line (Fig 11A) 641
In both cases we found the expression of three of the four analyzed genes to 642
be differentially regulated in the two genetic backgrounds suggesting that the 643
GATAs can cross-regulate their gene expression and supporting again the 644
hypothesis that the GATAs have a repressive activity on the transcription of 645
the other GATA genes Further support for such a hypothetical cross-646
regulation came from a ChIP-seq analysis with GNL where we observed a 647
strong binding of GNL when expressed from its own promoter to the three 648
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
22
GATA gene loci GNC GNL and GATA17 (Fig 11B) Additional studies are 649
needed to disentangle the interplay of the different LLM-domain B-GATAs in 650
the control of plant growth but also in the mutual transcriptional control of the 651
different GATA gene family members to get a full understanding of their 652
biochemical and biological function during plant growth and development 653
654
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
23
MATERIAL AND METHODS 655
Biological material All experiments were performed in Arabidopsis thaliana 656
ecotype Columbia Insertion mutants for GNC (SALK_001778) and GNL 657
(SALK_003995) and their double mutant were previously described (Richter 658
et al 2010) Insertion mutants for GATA15 (SAIL_618B11 and 659
WiscDsLox471A10) GATA16 (SALK_021471) GATA17 (SALK_101994 and 660
SALK_049041) and GATA17L (SALK_026798) were obtained from the 661
Nottingham Arabidopsis Stock Center Homozygous single and higher order 662
mutant combinations were isolated from segregating populations by PCR-663
based genotyping In the context of this analysis it became apparent that the 664
insertion position for one GATA15 allele (SAIL_618B11) was misannotated 665
and that the correct position of this insertion was downstream of the genes 3-666
untranslated region A list of genotyping primers is provided in Supplemental 667
Table S4 In addition the previously described mutants phyA-211 phyB-9 668
(Reed et al 1994) cry1 cry2 (Mockler et al 1999) and pifq (pif1 pif3 pif4 669
pif5) were used (Leivar et al 2008) 670
671
Protein alignment and phylogeny LLM-domain B-GATA protein sequences 672
of different Brassicaceae genomes were identified in the ENSEMBL database 673
(httpplantsensemblorgindexhtml) Sequences were filtered for proteins 674
containing the LLM-domain and alignments were performed using the 675
ClustalW2 alignment tool (httpwwwebiacukToolsmsaclustalw2) Two 676
Brassica rapa genes Bra037421 and Bra035633 were not included in the 677
analysis because no expressed sequence tags could be detected for these 678
annotations and they may represent pseudogenes 679
(httpwwwplantgdborgBrGDB) The phylogenetic tree was generated with 680
MEGA606 (httpwwwmegasoftwarenet) using the Neighbor-joining method 681
and the bootstrap method with 1000 bootstrap replications as well as the 682
Jones-Taylor-Thornton model with gapsmissing data treatment set to 683
pairwise deletion based on an alignment of trimmed B-GATAs using the entire 684
B-GATA DNA-binding domain and the C-termini with the LLM-domain (Fig 685
S2) 686
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
24
687
Physiological assays Unless otherwise stated all plants were cultivated on 688
sterile 12 MS medium without sugar under continuous white light (120 micromol 689
m-2 s-1) For hypocotyl length measurements seedlings were grown for 5 days 690
in white light (80 micromol m-2 s-1) far-red light (weak 035 micromol s-1 m-2 strong 691
06 micromol s-1 m-2) red light (weak 72 micromol s-1 m -2 strong 11 micromol s-1 m -2) 692
and blue light (weak 425 micromol s-1 m-2 strong 10 micromol s-1 m -2) Hypocotyl 693
length was measured from scanned seedlings using the NIH ImageJ software 694
Chlorophyll measurements were performed with 14 day-old plants and 695
chlorophyll content was determined as previously described and normalized 696
to the chlorophyll content of the wild type seedlings grown in the same 697
conditions (Inskeep and Bloom 1985) To visualize chloroplast accumulation 698
and auto-fluorescence pictures of seven day-old seedlings were taken with 699
an Olympus FV1000 confocal microscope (Olympus Hamburg Germany) 700
with an excitation of 405 nm and a detection from 631 to 729 nm The 701
experiments were repeated three times with comparable outcomes the result 702
of one representative experiment is shown For flowering time analyses 703
plants were grown on soil in 100 micromol m-2 s-1 in 16 hrs light8 hrs dark long 704
day-conditions or in 130 micromol m-2 s-1 8 hrs light16 hrs short day-conditions 705
For CK treatments 10 day-old seedlings were pre-incubated for four hours in 706
liquid 12 MS medium and treatments were started by adding hormone-707
containing medium to reach a final concentration of 10 microM 6-BA (6-708
benzylaminopurin) To examine the effects of CK on hypocotyl elongation 709
growth seedlings were grown in the dark for five days on medium containing 710
005 or 01 microM 6-BA or a corresponding mock solution Silique angles were 711
determined as previously described (Besnard et al 2014) To this end a 712
round gauge was built in such a way that it could be moved up the main 713
inflorescence to measure the angles of consecutive siliques The experiment 714
was repeated in two rounds and both datasets were combined Senescence 715
assays were performed as previously described (Weaver and Amasino 2001) 716
In brief detached leaves number three and five of 21 day-old light-grown 717
plants were floated on liquid 12 MS in absolute darkness containing either 718
005 or 01 microM BA or a mock solution After four days chlorophyll was 719
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
25
extracted as described above Alternatively whole plants were transferred 720
into darkness for four days chlorophyll was determined as described above 721
and total protein concentration was determined using a Bradford assay To 722
assess the branching phenotype of gata mutants the numbers of branches of 723
at least 05 cm length were determined from nodes in the rosette and from 724
cauline nodes as well as those of vegetative buds (leaf bearing nodes) along 725
the shoot For scanning electron microscopy of floral buds samples were 726
mounted on pre-frozen platforms and pictures were taken with a TM-3000 727
table top scanning electron microscope (Hitachi Krefeld Germany) Numbers 728
of floral organs were counted at the time of anthesis (stage 13) This 729
measurement was repeatedly performed with all plants after new flowers had 730
formed Siliques were harvested from different plants from a comparable 731
section of the main inflorescence Length and seed number was determined 732
for each silique separately To determine flowering plants were monitored on 733
a daily basis for the presence of a visible inflorescence bud (time to bolting) or 734
petals (flowering time) The numbers of rosette and cauline leaves after 735
bolting were counted 736
737
Microarray analyses For microarray analyses 14 day-old plants grown in 738
continuous white light (120 μmol mminus2 sminus1) were treated for 60 min with CK (20 739
microM 6-BA) or a corresponding mock solvent control treatment Total RNA was 740
extracted with the NucleoSpin RNA Plant Kit (Machery-Nagel Duumlren 741
Germany) and 150 ng total RNA was labeled with Cy3 using the Low Input 742
Quick Amp Labeling Protocol (Agilent Technologies Boumlblingen Germany) 743
Three biological replicate samples were prepared for each genotype and 744
Arabidopsis arrays (V4 design ID 21169 Agilent Technologies Boumlblingen 745
Germany) were hybridized at 65 degC for 17 hrs in rotating hybridization 746
chambers (Agilent Technologies Boumlblingen Germany) Subsequently the 747
arrays were washed and scanned using an Agilent Microarray Scanner 748
(Agilent Technologies Boumlblingen Germany) Total RNA and probe quality 749
were controlled with a Bioanalyzer 2100 (Agilent Technologies Boumlblingen 750
Germany) Raw data were extracted using the Feature Extraction software 751
v10731 (Agilent Technologies) Raw data files were imported into 752
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
26
GeneSpring GX (v 12) and normalized choosing the scale-to-median and 753
baseline-to-median algorithms Data were then subjected to an ANOVA 754
analysis (P lt 005) with an S-N-K post-hoc test and filtered for genes with fold 755
expression differences to the respective wild type controls as specified 756
(Supplemental Table S2) Microarray data were deposited as GSE71828 to 757
the Gene Expression Omnibus database (wwwncbinlmnihgovgeo) 758
759
Quantitative real time-PCR To measure transcript abundance total RNA 760
was extracted with RNeasy Plant Mini Kit (Qiagen Hilden Germany) 2 microg 761
total RNA was reverse transcribed with an oligo(dT) primer and M-MuLV 762
Reverse Transcriptase (Fermentas St Leon-Rot Germany) The transcript 763
levels were detected using a CFX96 Real-Time System Cycler with iQ SYBR 764
Green Supermix (Biorad Freiburg Germany) The results were normalized to 765
PP2A (PROTEIN PHOSPHATASE SUBUNIT2A AT1G13320) in red and far- 766
red light and to ACT2 (ACTIN2 AT3G18780) in blue light Expression in the 767
dark samples was set to one Normalization for the hormone treatments was 768
performed with ACT8 (ACTIN8 AT1G49240) Primers for qRT-PCRs are 769
listed in Supplemental Table S4 770
771
ChIP-seq For ChIP three biological replicate samples (2 g tissue) of 10 day-772
old pGNLGNLHA gnc gnl or gnc gnl control plants grown on GM medium 773
under constant white light were fixed for 20 min in 1 formaldehyde The 774
samples were subsequently processed as previously described using anti-HA 775
antibodies (Abcam Cambridge UK)(Kaufmann et al 2010) and prepared for 776
DNA sequencing with an Illumina MiSeq sequencer The data from this 777
analysis are accessible under PRJNA288918 at NCBI-SRA 778
(httpwwwncbinlmnihgovsra) Sequencing reads were then mapped to 779
the Arabidopsis genome (TAIR10) using SOAPv1 (Li et al 2008) and the 780
following settings 3 mismatches mapping to unique positions only no gaps 781
allowed iteratively trimming set to 41 - 50 Subsequent analysis for peak 782
identification was performed with CSAR retaining only peaks with an FDR lt 783
005 as statistically significant peaks (Muino et al 2011) ChIP-seq results for 784
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
27
GATA genes were independently verified using ChIP-PCR The primers for 785
ChIP-PCRs are listed in Supplemental Table S4 786
787
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
28
SUPPLEMENTAL MATERIALS 788
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within the 789
Brassicaceae 790
Figure S2 Sequence alignment of the GATA-domain and the LLM-domain 791
Figure S3 GATA insertion mutant analysis 792
Figure S4 Mature plant phenotypes of gata gene mutants 793
Figure S5 GATA gene expression in red light conditions is PIF-dependent 794
Figure S6 The effects of CK-induction in the two microarray data sets are 795
comparable 796
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 797
Figure S8 The effect of CK on hypocotyl elongation is not impaired in GATA 798
gene mutants 799
Figure S9 GATA gene dosage affects lateral inflorescence angles 800
Figure S10 Floral morphology and silique parameters are influenced in 801
GATA gene mutations 802
Figure S11 Quantitative analysis of flowering time in long and short day-803
conditions 804
Figure S12 GATA gene mutations alter flowering time in long day and short 805
day conditions 806
807
Supplemental Table S1 Differentially expressed genes in GATA gene 808
mutants 809
Supplemental Table S2 Differentially expressed genes in GATA gene 810
mutants after CK-treatment 811
Supplemental Table S3 Abundance of CK-regulated genes after CK-812
treatment in the wild type and GATA gene mutants 813
Supplemental Table S4 Primers used in this study 814
815
816
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
29
REFERENCES 817
Behringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) 818
Functional diversification within the family of B-GATA transcription 819
factors through the leucine-leucine-methionine domain Plant Physiol 820
166 293-305 821
Behringer C Schwechheimer C (2015) B-GATA transcription factors - 822
insights into their structure regulation and role in plant development 823
Front Plant Sci 6 90 824
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P 825
Rozier F Mirabet V Legrand J Laine S Thevenon E Farcot E 826
Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y 827
Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014) 828
Cytokinin signalling inhibitory fields provide robustness to phyllotaxis 829
Nature 505 417-421 830
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE 831
Loraine A Kieber JJ (2013) Identification of cytokinin-responsive 832
genes using microarray meta-analysis and RNA-Seq in Arabidopsis 833
Plant Physiol 162 272-294 834
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic 835
analysis of Arabidopsis GATA transcription factor gene family reveals a 836
nitrate-inducible member important for chlorophyll synthesis and 837
glucose sensitivity Plant J 44 680-692 838
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon 839
M Kieber JJ Schaller GE (2012) Functional characterization of the 840
GATA transcription factors GNC and CGA1 reveals their key role in 841
chloroplast development growth and division in Arabidopsis Plant 842
Physiol 160 332-348 843
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W 844
Audenaert D Van Campenhout J Overvoorde P Jansen L 845
Vanneste S Moller B Wilson M Holman T Van Isterdael G 846
Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers 847
D Bennett MJ Beeckman T (2010) A novel AUXIAA28 signaling 848
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
30
cascade activates GATA23-dependent specification of lateral root 849
founder cell identity Curr Biol 20 1697-1706 850
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin 851
signalling and crosstalk Development 140 1373-1383 852
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates 853
APETALA1 function in the establishment of determinate floral 854
meristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845 855
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev 856
Plant Biol 63 353-380 857
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B 858
in NN-dimethylformamide and 80 acetone Plant Physiol 77 483-859
485 860
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V 861
Serrano-Mislata A Madueno F Krajewski P Meyerowitz EM 862
Angenent GC Riechmann JL (2010) Orchestration of floral initiation 863
by APETALA1 Science 328 85-89 864
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro 865
EM Fukaki H Ohta H Sugimoto K Masuda T (2012) Regulation of 866
root greening by light and auxincytokinin signaling in Arabidopsis 867
Plant Cell 24 1081-1095 868
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different 869
cytokinin-regulated transcription factor genes of Arabidopsis thaliana 870
alters plant growth and development J Plant Physiol 168 1320-1327 871
Leivar P Monte E (2014) PIFs systems integrators in plant development 872
Plant Cell 26 56-78 873
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH 874
(2008) Multiple phytochrome-interacting bHLH transcription factors 875
repress premature seedling photomorphogenesis in darkness Curr 876
Biol 18 1815-1823 877
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide 878
alignment program Bioinformatics 24 713-714 879
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
31
Mara CD Irish VF (2008) Two GATA transcription factors are downstream 880
effectors of floral homeotic gene action in Arabidopsis Plant Physiol 881
147 707-718 882
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of 883
Arabidopsis cryptochromes and phytochrome B in the regulation of 884
floral induction Development 126 2073-2082 885
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) 886
ChIP-seq Analysis in R (CSAR) An R package for the statistical 887
detection of protein-bound genomic regions Plant Methods 7 11 888
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) 889
Characterization of a unique GATA family gene that responds to both 890
light and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 891
71 1557-1560 892
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) 893
The GATA factor HANABA TARANU is required to position the 894
proembryo boundary in the early Arabidopsis embryo Dev Cell 19 895
103-113 896
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A 897
and phytochrome B have overlapping but distinct functions in 898
Arabidopsis development Plant Physiol 104 1139-1149 899
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of 900
transcription factors in Arabidopsis and rice Plant Physiol 134 1718-901
1732 902
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive 903
interactions between SOC1 and the GATAs GNC and GNLCGA1 in 904
the control of greening cold tolerance and flowering time in 905
Arabidopsis Plant Physiol 162 1992-2004 906
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-907
type transcription factors GNC and GNLCGA1 repress gibberellin 908
signaling downstream from DELLA proteins and PHYTOCHROME-909
INTERACTING FACTORS Genes Dev 24 2093-2104 910
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
32
Richter R Behringer C Zourelidou M Schwechheimer C (2013) 911
Convergence of auxin and gibberellin signaling on the regulation of the 912
GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc 913
Natl Acad Sci U S A 110 13192-13197 914
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl 915
elongation in Arabidopsis seedlings are independent and additive 916
Plant Physiol 108 1423-1430 917
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G 918
Wang X Freeling M Pires JC (2012) Altered patterns of fractionation 919
and exon deletions in Brassica rapa support a two-step model of 920
paleohexaploidy Genetics 190 1563-1574 921
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I 922
Cheng F Huang S Li X Hua W Wang J Wang X Freeling M Pires 923
JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG 924
Park BS Weisshaar B Liu B Li B Liu B Tong C Song C Duran C 925
Peng C Geng C Koh C Lin C Edwards D Mu D Shen D 926
Soumpourou E Li F Fraser F Conant G Lassalle G King GJ 927
Bonnema G Tang H Wang H Belcram H Zhou H Hirakawa H 928
Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J 929
Li J Xu J Deng J Kim JA Li J Yu J Meng J Wang J Min J 930
Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M 931
Links MG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai 932
Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S 933
Sato S Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X 934
Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y 935
Wang Y Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa 936
Genome Sequencing Project C (2011) The genome of the 937
mesopolyploid crop species Brassica rapa Nat Genet 43 1035-1039 938
Weaver LM Amasino RM (2001) Senescence is induced in individually 939
darkened Arabidopsis leaves but inhibited in whole darkened plants 940
Plant Physiol 127 876-886 941
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
33
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ 942
Jackson DP (2010) A conserved mechanism of bract suppression in 943
the grass family Plant Cell 22 565-578 944
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription 945
repressor HANABA TARANU controls flower development by 946
integrating the actions of multiple hormones floral organ specification 947
genes and GATA3 family genes in Arabidopsis Plant Cell 25 83-101 948
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz 949
EM (2004) HANABA TARANU is a GATA transcription factor that 950
regulates shoot apical meristem and flower development in Arabidopsis 951
Plant Cell 16 2586-2600 952
953
ACKNOWLEDGMENTS 954
CS would like to thank Eilon Shani and Daniel Chamovitz Department of 955
Molecular Biology and Ecology of Plants for hosting CS at Tel Aviv 956
University during manuscript preparation QLR EB and CS would like to 957
thank Jutta Elgner for her excellent technical support 958
959
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
34
FIGURE LEGENDS 960
Figure 1 LLM-domain B-GATAs redundantly control greening in 961
Arabidopsis thaliana A Domain architecture of short and long LLM-domain 962
B-GATAs based on the alignment of family members from Arabidopsis 963
thaliana Protein regions with restricted sequence conservation or gaps in the 964
alignment are depicted as lines regions with low (le 50) sequence similarity 965
are depicted as grey boxes regions with high (ge 80) sequence similarity as 966
black boxes Scale bar = 30 amino acids in a conserved part of the protein B 967
MUSCLE alignment with sequence logo of the B-GATA domain and the LLM-968
domain of the Arabidopsis thaliana LLM-domain B-GATAs C Schematic 969
representation of the gene models of the six Arabidopsis LLM-domain B-970
GATA genes with exons (boxes) and introns (lines) The 5rsquo-UTR (untranslated 971
region) of GNL and the 3-UTR of GATA16 were previously unknown and 972
derived from RNA sequencing data available in the laboratory The 973
sequences coding for the GATA DNA-binding domain and the LLM-domain 974
are represented as black and blue boxes respectively The T-DNA insertion 975
positions are shown The position in brackets of the GATA15 insertion refers 976
to the original misannotation of this insertion (wwwsignalsalkedu) D 977
Photograph of 14 day-old plants with the genotypes as specified in 978
comparison to the Columbia-0 (Col-0) wild type control E Result of the 979
quantification of chlorophyll A and B from different gata single and complex 980
mutants The experiment was performed in two rounds as indicated by the 981
lines above the genotypes to reduce the complexity of the experiment the 982
Col-0 wild type reference was included in both rounds F Representative 983
photographs of seven day-old seedlings (upper panel) and confocal images 984
(lower panel) of these seedlings to visualize differential chloroplast 985
accumulation in these mutants 986
Figure 2 GATA gene expression in far-red red and blue light 987
conditions A ndash C Averages and standard errors of three replicate qRT-PCR 988
analyses of four day-old dark-grown seedlings after transfer to (A) far-red 989
(035 micromol s-1 m-2) (B) red (72 micromol s-1 m -2) and (C) blue light (425 micromol s-1 990
m-2) phyA phyB and cry1 cry2 light receptor mutants were used to control the 991
specificity of the respective treatment and to control for background gene 992
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
35
expression Gene expression data were normalized to the transcript 993
abundance detected in the dark sample of the respective genotype Studentrsquos 994
t-test for selected time points p le 005 p le 001 p le 0001 ns = not 995
significant 996
Figure 3 GATA gene expression is induced by cytokinin Averages and 997
standard errors of three replicate qRT-PCR analyses of ten day-old seedlings 998
that had been treated for the times indicated with 10 microM 6-BA Expression 999
changes for CKX4 are shown to verify the effectiveness of the hormone 1000
treatment The expression values for each time point were normalized to 1001
those of an untreated mock sample The fold change is shown relative to time 1002
point 0 which was set to 1 Studentrsquos t-test for selected time points p le 1003
005 p le 001 p le 0001 ns = not significant 1004
Figure 4 GATA factors contribute to hypocotyl elongation A ndash E 1005
Representative photographs as well as F ndash M averages and standard errors 1006
from hypocotyl measurements of five day-old seedlings (n ge 50) grown in 1007
white light (A and F) weak and strong red light (B H I) weak and strong far-1008
red light (C J K) weak and strong blue light (D L and M) and in the dark (E 1009
and G) Scale bar = 2 mm Studentrsquos t-test datasets with no statistical 1010
difference fall in one group and were labeled accordingly 1011
Figure 5 Differential basal gene expression in gata mutants A 1012
Heatmaps of differentially expressed genes from ten day-old light-grown Col-0 1013
seedlings and the gnc gnl quintuple and triple mutants The experiment was 1014
performed in two rounds with two independent wild type controls referred to 1015
as Col-0 (1) and Col-0 (2) Down-regulated and up-regulated genes are 1016
shown in the upper and lower panels respectively B Venn diagrams 1017
showing the overlap in differential gene expression for down- and up-1018
regulated genes between the three different gata mutant backgrounds using 1019
the respective Col-0 wild type control as a reference Gene expression 1020
identities and data are shown in Supplemental Table S1 1021
Figure 6 Cytokinin-responsive gene expression is impaired in gata 1022
mutants A and B Heatmaps and Venn diagrams of differentially expressed 1023
genes from Col-0 and the gata mutants after 60 min CK treatment The 1024
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
36
experiment was performed in two rounds with two independent wild type 1025
controls referred to as Col-0 (1) and Col-0 (2) Down-regulated and up-1026
regulated genes are shown in the upper and lower panels respectively Venn 1027
diagrams showing the overlap in differential gene expression for genes that 1028
were down- and up-regulated following CK treatment in the respective Col-0 1029
wild type and their expression in the respective gata mutants Gene 1030
expression identities and data are shown in Supplemental Table S2 1031
Figure 7 Phyllotaxis patterns are impaired in GATA gene mutants A 1032
Representative photographs of mature inflorescences Arrowheads point at 1033
events of aberrant silique positioning B Measurement of silique angles along 1034
the primary inflorescence of representative wild type and quintuple mutant 1035
plants Note the particularly strong deviation from the ideal silique angle of 1036
1375deg at the basis of the inflorescence C Frequency distribution of silique 1037
divergence angles in wild type and gata triple as well as quintuple mutant 1038
plants Arrowheads mark examples for instances were mutant angles strongly 1039
deviate from wild type angles D Overview over the number of siliques with a 1040
non-canonical angle as observed in the wild type and GATA gene mutants 1041
Figure 8 Cytokinin-effects on senescence are impaired in gata mutants 1042
A ndash E Representative photographs of leaves from 21 day-old plants Leaves 1043
number three and five were detached and kept in the dark for four days in 1044
liquid medium to monitor senescence in the presence and absence of the 1045
cytokinin 6-benzylaminopurin (BA) Scale bar = 1 cm F Averages and 1046
standard deviation after quantification of chlorophyll abundance of the leaves 1047
shown in (A ndash E) (n = 3) Studentrsquos t-test p le 005 p le 001 p le 0001 1048
ns = not significant G Representative photographs of 33 day-old plants that 1049
had transferred from the light to the dark for four days Scale bar = 1 cm H 1050
and I Averages and standard deviations after quantification of chlorophyll 1051
measurements (H) and total protein determination (I) of plants shown in (G) n 1052
ge 3 Studentrsquos t-test was performed for dark samples datasets with no 1053
statistical difference fall in one group and were labeled accordingly 1054
Figure 9 GATA gene mutants have altered branching patterns and 1055
branch angles A Representative photographs of seven week-old Col-0 and 1056
quintuple mutant plants Arrowheads indicate positions of second order lateral 1057
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
37
branch formation (white) and first order branches from nodes in the rosette 1058
(blue) Scale bar = 5 cm B C and F Quantitative analysis of branch 1059
numbers (first order branch from cauline leaves first o br cauline second 1060
order branches second o br and first order branches from the rosette first o 1061
br rosette) (B) plant height (C) and vegetative bud formation along the shoot 1062
(F) in the wild type and gata mutant backgrounds n = 15 Studentrsquos t-test 1063
datasets with no statistical difference fall in one group and were labeled 1064
accordingly D and E Photographs (D) and scanning electron micrographs 1065
(E) of floral meristems and siliques formed in the axils of second order lateral 1066
branches of quintuple mutants which are typically not found in the wild type 1067
Scale bar = 1 mm F Quantitative analysis of branch angles measured on five 1068
week-old Arabidopsis plants n = 19 Studentrsquos t-test p le 005 p le 001 1069
p le 0001 ns not significant G Representative photographs of lateral 1070
inflorescences branching from the primary inflorescence in the wild type The 1071
arrows indicate the angles measured for the analysis shown in (F) Scale bar 1072
= 1 cm 1073
Figure 10 gata mutants have impaired floral morphology and flowering 1074
time A Phenotypic analysis of sepal and petal numbers in the wild type and 1075
the gata gene mutant backgrounds Percentages indicate the penetrance of 1076
the displayed floral phenotype The wild type flower phenotype represents 1077
about 98 of all flowers analyzed the frequency of aberrant flowers is similar 1078
to the one of the gnc gnl mutant B and C Representative photographs (side 1079
views and top views) of wild type and gata gene mutants grown for five weeks 1080
in long-day (16 hrs light8hrs dark) (B) or for ten weeks in short-day (8 hrs 1081
light16 hrs dark) conditions (C) Scale bar = 5 cm 1082
Figure 11 GATA gene expression is cross-regulated in gnc gnl mutants 1083
and GNLox transgenic lines A Result from qRT-PCR analyses of the 1084
GATA genes GATA15 GATA16 GATA17 and GATA17L in the gnc gnl 1085
mutant and GNLox background Gene expression data were normalized to 1086
the transcript abundance detected in the Col-0 wild type B Read coverage 1087
over the six LLM-domain B-GATA genes and 3 kb upstream region after 1088
chromatin immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl 1089
transgenic line (blue line) and the gnc gnl mutant (red line) followed by next 1090
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
38
generation sequencing GATA gene models and where applicable the gene 1091
models of the neighboring genes are indicated in the figure different GATA-1092
sequence containing motifs are indicated below the gene models C Result 1093
from qRT-PCR analyses for selected amplicons (grey boxes) upstream from 1094
the GNC GNL and GATA17 gene promoters after chromatin 1095
immunoprecipitation of GNLHA from a pGNLGNLHA gnc gnl transgenic line 1096
and the gnc gnl mutant Studentrsquos t-tests p le 005 p le 001 p le 0001 1097
ns = not significant 1098
1099
SUPPLEMENTAL FIGURE LEGENDS 1100
Figure S1 Evolutionary conservation of LLM-domain B-GATAs within 1101
the Brassicaceae Phylogenetic tree of the LLM-domain B-GATAs from 1102
Brassicaceae species Arabidopsis thaliana (At) Arabidopsis lyrata (Al) 1103
Capsella rubella (Cr) Eutrema salsugineum (Es) and Brassica rapa (Br) The 1104
underlying alignment is shown in Fig S2 Species identities are color-coded 1105
for ease of viewing bootstrap values are shown at nodes The tree is drawn 1106
to scale with branch lengths in the same units as those of the evolutionary 1107
distances used to infer the phylogenetic tree The evolutionary distances were 1108
computed using the JTT matrix-based method and are in the units of the 1109
number of amino acid substitutions per site 1110
Figure S2 Sequence alignment of the GATA-domain and the LLM-1111
domain Pretty box sequence alignment (httpwwwebiacukTools 1112
msaclustalw2) of the Brassicaceae GATA factors used for the phylogenetic 1113
analysis shown in Figure 1 Sequence conservation is represented by a 1114
sequence logo above the respective alignments Black boxed residues 1115
indicate completely conserved amino acids different shades of grey indicate 1116
residues that are highly conserved between the aligned sequences with 1117
darker grey reflecting a higher degree of conservation (dark grey 80-100 1118
light grey 60-80 white less than 60) 1119
Figure S3 GATA insertion mutant analysis A Schematic representation 1120
of the gene models of the six Arabidopsis LLM-domain B-GATA genes with 1121
exons (boxes) and introns (lines) The sequences coding for the GATA DNA-1122
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
39
binding domain and the LLM-domain are represented as black and blue boxes 1123
respectively The T-DNA insertion positions are shown The position in 1124
brackets of the GATA15 insertion refers to the original misannotation of this 1125
insertion (wwwsignalsalkedu) The image is identical to the one shown in 1126
Figure 1C except that the positions of the primer pairs used for sqPCR (black 1127
arrowheads) and qRT-PCR (green and red arrowheads) were added B 1128
Results from qRT-PCR analyses (left panels fold change refers to normalized 1129
fold change) and sqRT-PCR analyses (right panels) with RNA prepared from 1130
seven day-old Col-0 wild type seedlings and gata gene mutants In the sqRT-1131
PCR experiment genomic DNA (gDNA) was used to control for contamination 1132
A reaction mix with water (H2O) was used to control for purity of the reaction 1133
components The ACTIN2 (ACT2) gene was used as a reference gene for the 1134
sqRT-PCR 1135
Figure S4 Mature plant phenotypes of GATA gene mutants 1136
Representative photographs of Arabidopsis plants at 25 days after 1137
germination (dag) 28 dag 31 dag and 35 dag Please note that the 1138
chlorophyll accumulation defect is maintained in the quintuple mutant but lost 1139
in the other mutants Scale bar = 5 cm 1140
Figure S5 GATA gene expression in red light conditions is PIF-1141
dependent Averages and standard errors of three replicate qRT-PCR 1142
analyses of four day-old dark-grown wild type and pifq seedlings after transfer 1143
to red (72 micromol s-1 m -2) light Studentrsquos t-test for the 12 hr time point gnc gnl 1144
vs Col p le 005 quintuple vs Col p le 001 triple vs Col ns = not 1145
significant 1146
Figure S6 The effects of CK-induction in the two microarray data sets 1147
are comparable A Graphic representation of the CK-induction of ARR 1148
genes from Arabidopsis thaliana in the two microarray data sets reveals that 1149
CK-induction was comparable between the two Col-0 wild type controls as 1150
well as in the different GATA gene mutants B Venn diagrams comparing the 1151
CK-induction (two-fold induction) in the two wild type samples Col-0 (1) and 1152
Col-0 (2) with the ldquogolden listrdquo of previously identified CK-regulated genes 1153
See Supplemental Table S3 for a list of all CK-regulated genes from the 1154
ldquogolden listrdquo 1155
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
40
Figure S7 CK cannot efficiently induce greening in GATA gene mutants 1156
Result of the quantification of chlorophyll A and B from 14 day-old wild type 1157
and different gata mutant plants grown in the absence and presence of 5 nM 1158
6-BA Studentrsquos t-tests p le 001 ns = not significant 1159
Figure S8 The effect of CK on hypocotyl elongation is not impaired in 1160
GATA gene mutants Result from a hypocotyl elongation assay with five day-1161
old dark-grown seedlings Studentrsquos t-tests not significant for all comparisons 1162
between different genotypes subjected to identical treatments (n gt 25) 1163
Figure S9 GATA gene dosage affects lateral inflorescence angles 1164
Representative photographs of lateral branches branching off the primary 1165
inflorescences in the wild type GATA gene mutants and overexpressors 1166
Scale bar = 1 cm 1167
Figure S10 Floral morphology and silique parameters are influenced in 1168
GATA gene mutations A Quantitative phenotypic analysis of sepal and 1169
petal numbers in the wild type and the GATA gene mutant backgrounds B 1170
Photographs of representative mature siliques from the different genetic 1171
backgrounds C and D Quantitative analysis of seed number per silique (C) 1172
and silique length (D) Datasets with no statistical difference fall in one group 1173
and were labeled accordingly (n ge 57) 1174
Figure S11 Quantitative analysis of flowering time in long and short 1175
day-conditions A - D Quantitative analysis of flowering based on leaf 1176
number counts or days to bolting of the long day and short day-grown plants 1177
as shown in Figure 10B and C n = 20 Studentrsquos t-test datasets with no 1178
statistical difference fall in one group and were labeled accordingly 1179
Figure S12 GATA gene mutations alter flowering in long day and short 1180
day conditions Representative photographs of A five week-old plants 1181
grown in long day conditions or B ten week-old plants grown in short day-1182
conditions to demonstrate the acceleration of flowering in long day-grown 1183
mutant plants and the delay in flowering in mutants grown in short days 1184
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
C D
E
Chl
orop
hyll
[]
aa
b bc
d
Col-0
Col-0
gnc gn
l
gata1
5-1 (G
ATA15)
gata1
5-2ga
ta16
gata1
7-1
gata1
7-2
gata1
7l0
20406080
100120
GATA15
gata1
6
gnc g
nl ga
ta17 g
ata17
l
gata1
7 gata
17ltrip
le
gnc g
nl
gnc g
nl GATA
15 ga
ta16
quint
uple
Col-0
Col-0
gnc gnl
gnc gnl
triple
triple
quintuple
quintuple
GNLox
GNLox
quintuple
gncgnl
tripleGATA15gata16gata17gata17l
gncgnlgata17gata17l
gncgnlGATA15gata16
GATA15gata16
gata17gata17l
Col-0
F
short B-GATA
long B-GATA
short B-GATA
long B-GATA
LLM-domainGATA-domainA
B
Figure 1_Ranftl_et_al
LLM-domainGATA-domain
GATA17L GATA17 GATA15 GATA16 GNL GNC
GNC
GNL
GATA15
GATA16
GATA17
GATA17L
gnc (SALK_001778)
gnl (SALK_003995)
gata15-1 (GATA15 SAIL_618B11)gata15-2 (WiscDsLox471A10)
gata16-1 (SALK_021471)
gata17-1 (SALK_101994) gata17-2 (SALK_049041)
gata17l-1 (SALK_026798)
( )
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
far-red red blue
Figure 2_Ranftl_et_alA B C
Nor
mal
ized
fold
cha
nge
020406080
100120 GNL GNL
Nor
mal
ized
fold
cha
nge
0
20
40
60
Nor
mal
ized
fold
cha
nge
0
40
80
120
160 GNLCol-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GNC
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
dark 30 60 12
018
0da
rk 30 60 120
180
dark 30 60 12
018
0
GNC
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge GNC Col-0
cry1cry2 Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 GATA15
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA15 Col-0cry1cry2
Col-0phyA phyB
Col-0phyA phyB
ns
time [min] time [min]time [min]
Col-0cry1cry2
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge GATA16 GATA16
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5N
orm
aliz
ed fo
ld c
hang
e GATA16Col-0phyA phyB
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17 GATA17
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17 Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
GATA17LGATA17L
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
0
1
2
3
4
5
Nor
mal
ized
fold
cha
nge
Col-0phyA phyB
GATA17L Col-0cry1cry2
Col-0phyA phyB
time [min] time [min]time [min]
ns
ns ns
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
BA BA BA
BA
BA
BA BA
GNC
143x
148x 155x 157x
289x
136x
GNL GATA15
GATA16 GATA17 GATA17L
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
00
10
20
30
00
04
08
12
16
00
04
08
12
16
00
04
08
12
16
00
10
2030
40
50
00
04
08
12
16
00
04
08
1216
20
Figure 3_Ranftl_et_al
time [min] time [min]time [min]
0 60 120
180
240 0 60 12
018
024
0 0 60 120
180
240
time [min] time [min]
0 60 120
180
240
time [min]
time [min]
CKX4
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
Nor
mal
ized
fold
cha
nge
ns
384x
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 gnc gnl triple quint
triple quint
triple quint
pifq GNLox
Col-0 gnc gnl pifq GNLox
Col-0 gnc gnl pifq GNLox
A
B
C
red
light
whi
te li
ght
far-r
ed li
ght
Figure 4_Ranftl_et_al
Col-0 gnc gnl triple quint pifq GNLox
D
E
Col-0 gnc gnl triple quint pifq GNLox
blue
ligh
tda
rk
0
4
8
12
16 a
b
a
a ab b
c
dH
Ga
b
c
a
b
c
d
Leng
th[m
m]
0
2
6
8
I
Leng
th[m
m]
0
2
6
8
L
M
Leng
th[m
m]
Leng
th[m
m]
0
2
4
6
8
Leng
th[m
m]
red (weak)
red (strong)
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Col-0
gnc g
nl
quint
uple
triple pif
q
GNLox
Leng
th[m
m]
0
2
4
6
0
2
4
6
0
2
4
6
a bc c de
f
F white light J
K
Leng
th[m
m]
Leng
th[m
m]
0
2
4
4
6
8
b
c
dfar-red (weak) blue (weak)
dark far-red (strong) blue (strong)
c
d
d
a a
a
bc a b
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (1
)gn
c gn
lqu
intu
ple
Col
-0 (2
)tri
ple
Figure 5_Ranftl_et_al
-15 00 15
190
1880
87 828
187
211
542
11
89
50
triple (323)
quintuple (4722)
triple (278)
gnc gnl(294)
gnc gnl(2458)
quintuple (976)
down-regulated entities
up-regulated entities
531 2711
6
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col
-0 (2
)tri
ple
0020 -20
60 min BA
Col-0 (2)(188)
triple (143)
Col-0 (2)(282)
triple(367)
105
104 178 189
83 60
down-regulated entities
up-regulated entities
down-regulated entities
up-regulated entities
Figure 6_Ranftl_et_alC
ol-0
(1)
gnc
gnl
quin
tupl
e
60 min BA
0020 -20
807
110
115
155
16
77
317
81
14
22
73
139
100
39
Col-0 (1)(996)
gnc gnl (218)
quintuple (504)
Col-0 (1)(365)
gnc gnl (393)
quintuple (281)
A B
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Genotype non-canonical angle n (plants) n (siliques)
Freq
uenc
y
Freq
uenc
y
Col-0quintuple
Col-0triple
0100200300
0100200300
50 010 15 20 25 30 5 10 15 20 25 30
Col-0 quintuple
A gnc gnl triple quintuple Col-0
B
Angl
e [deg]
Divergence angle Divergence angle
Silique
02468
1012141618
02468
1012141618
0 100 200 3000 100 200 300
Figure 7_Ranftl_et_al
Angl
e [deg]
SiliqueC
DCol-0 2044 19 450gnc gnl 2058 23 520triple 3065 20 522quintuple 2953 22 550
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
triplegnc gnl quintuple GNLox
Col-0 gnc gnl triple quintuple GNLox0
1
2
3
4
5
6
70 d dark 5 d dark 0 microM BA5 d dark 005 microM BA 5 d dark 01 microM BA
Chl
orop
hyll
in micro
gm
g FW
F
nsns
ns
nsns ns
Col-0
gnc g
nltrip
le
quint
uple
GNLox
Col-0
gnc g
nltrip
le
quint
uple
GNLox
0004081216
Tota
l pro
tein
[microg
mg]
00
04
08
12
16 light dark
Chl
orop
hyll
[microg
mg]
HCol-0
Col
-0gn
c gn
ltri
ple
quin
tupl
eG
NLo
x
0 d dark 5 d dark
mock 005 microM BA 01 microM BA
A
B
C
D
E
G
Figure 8_Ranftl_et_al
I light dark
a b ab
c a a ab
a
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Col-0 quintuple
0
10
20
30
40
50
Plan
t hei
ght [
cm]
Num
ber o
f veg
etat
ive
buds
Num
ber o
f bra
nche
s
012
3456 first o br cauline second o br first o br rosette
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6ga
ta17
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
gnc g
nltrip
le
gnc g
nl GATA
15 ga
ta16
gnc g
nl ga
ta17 g
ata17
l
quint
uple
Col-0
GATA15
gata1
6
gata1
7
gata1
7l
GATA15
gata1
6
gata1
7 gata
17l
A B
C
F
0
510
1520
25
30
a
a
a
a
a a aa
a a a a a
b bab
ab
a aa a a
b
b b
bb
b
c
c
c
c d eed c
dcd
cc
bc
d e
b ba
e
a
Col-0 quintuple quintupleD
Col-0 quintuple quintupleE
Figure 9_Ranftl_et_al
H
40
50
Col-0
gnc g
nltrip
leGNLo
x
quint
uple
60
70
80
Angl
e [deg]
G
ns
Col-0
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
00
05
10
15
20
25
GATA15 GATA16 GATA17 GATA17L
Col-0gnc gnlGNLox
Nor
mal
ized
fold
cha
nge
Figure 11_Ranftl_et_al
ns ns
TGATAG
AGATAA
TGATAA
AGATAG
IP with Anti-HA for gnc gnlIP with Anti-HA for pGNLGNLHA gnc gnl
GATA16250 bp
GATA17250 bp
GNC250 bp
GNL250 bp
AT3G06720
GATA15250 bp
AT3G06730GATA17L250 bp
ATG16143
A C
B
promoter GNC
50 bp 50 bpATG
00
02
04
06
08
10
12
00
02
04
06
08
10
00
02
04
06
08
101214
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
GN
LH
A en
richm
ent (
o
f Inp
ut)
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
pGNL
GNLHA
gnc
gnl
gnc g
nl
promoter GATA17
50 bpATG
promoter GNL
ATG
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Parsed CitationsBehringer C Bastakis E Ranftl QL Mayer KF Schwechheimer C (2014) Functional diversification within the family of B-GATAtranscription factors through the leucine-leucine-methionine domain Plant Physiol 166 293-305
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Behringer C Schwechheimer C (2015) B-GATA transcription factors - insights into their structure regulation and role in plantdevelopment Front Plant Sci 6 90
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Besnard F Refahi Y Morin V Marteaux B Brunoud G Chambrier P Rozier F Mirabet V Legrand J Laine S Thevenon E FarcotE Cellier C Das P Bishopp A Dumas R Parcy F Helariutta Y Boudaoud A Godin C Traas J Guedon Y Vernoux T (2014)Cytokinin signalling inhibitory fields provide robustness to phyllotaxis Nature 505 417-421
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bhargava A Clabaugh I To JP Maxwell BB Chiang YH Schaller GE Loraine A Kieber JJ (2013) Identification of cytokinin-responsive genes using microarray meta-analysis and RNA-Seq in Arabidopsis Plant Physiol 162 272-294
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bi YM Zhang Y Signorelli T Zhao R Zhu T Rothstein S (2005) Genetic analysis of Arabidopsis GATA transcription factor genefamily reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity Plant J 44 680-692
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chiang YH Zubo YO Tapken W Kim HJ Lavanway AM Howard L Pilon M Kieber JJ Schaller GE (2012) Functionalcharacterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development growth anddivision in Arabidopsis Plant Physiol 160 332-348
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
De Rybel B Vassileva V Parizot B Demeulenaere M Grunewald W Audenaert D Van Campenhout J Overvoorde P Jansen LVanneste S Moller B Wilson M Holman T Van Isterdael G Brunoud G Vuylsteke M Vernoux T De Veylder L Inze D Weijers DBennett MJ Beeckman T (2010) A novel AUXIAA28 signaling cascade activates GATA23-dependent specification of lateral rootfounder cell identity Curr Biol 20 1697-1706
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
El-Showk S Ruonala R Helariutta Y (2013) Crossing paths cytokinin signalling and crosstalk Development 140 1373-1383Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Han Y Zhang C Yang H Jiao Y (2014) Cytokinin pathway mediates APETALA1 function in the establishment of determinate floralmeristems in Arabidopsis Proc Natl Acad Sci U S A 111 6840-6845
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hwang I Sheen J Muller B (2012) Cytokinin signaling networks Annu Rev Plant Biol 63 353-380Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Inskeep WP Bloom PR (1985) Extinction coefficients of chlorophyll A and B in NN-dimethylformamide and 80 acetone PlantPhysiol 77 483-485
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kaufmann K Wellmer F Muino JM Ferrier T Wuest SE Kumar V Serrano-Mislata A Madueno F Krajewski P Meyerowitz EMAngenent GC Riechmann JL (2010) Orchestration of floral initiation by APETALA1 Science 328 85-89
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kobayashi K Baba S Obayashi T Sato M Toyooka K Keranen M Aro EM Fukaki H Ohta H Sugimoto K Masuda T (2012)Regulation of root greening by light and auxincytokinin signaling in Arabidopsis Plant Cell 24 1081-1095
Pubmed Author and Title wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
CrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kollmer I Werner T Schmulling T (2011) Ectopic expression of different cytokinin-regulated transcription factor genes ofArabidopsis thaliana alters plant growth and development J Plant Physiol 168 1320-1327
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E (2014) PIFs systems integrators in plant development Plant Cell 26 56-78Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Leivar P Monte E Oka Y Liu T Carle C Castillon A Huq E Quail PH (2008) Multiple phytochrome-interacting bHLH transcriptionfactors repress premature seedling photomorphogenesis in darkness Curr Biol 18 1815-1823
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li R Li Y Kristiansen K Wang J (2008) SOAP short oligonucleotide alignment program Bioinformatics 24 713-714Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mara CD Irish VF (2008) Two GATA transcription factors are downstream effectors of floral homeotic gene action in ArabidopsisPlant Physiol 147 707-718
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Mockler TC Guo H Yang H Duong H Lin C (1999) Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in theregulation of floral induction Development 126 2073-2082
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Muino JM Kaufmann K van Ham RC Angenent GC Krajewski P (2011) ChIP-seq Analysis in R (CSAR) An R package for thestatistical detection of protein-bound genomic regions Plant Methods 7 11
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Naito T Kiba T Koizumi N Yamashino T Mizuno T (2007) Characterization of a unique GATA family gene that responds to bothlight and cytokinin in Arabidopsis thaliana Biosci Biotechnol Biochem 71 1557-1560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nawy T Bayer M Mravec J Friml J Birnbaum KD Lukowitz W (2010) The GATA factor HANABA TARANU is required to position theproembryo boundary in the early Arabidopsis embryo Dev Cell 19 103-113
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reed JW Nagatani A Elich TD Fagan M Chory J (1994) Phytochrome A and phytochrome B have overlapping but distinctfunctions in Arabidopsis development Plant Physiol 104 1139-1149
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Reyes JC Muro-Pastor MI Florencio FJ (2004) The GATA family of transcription factors in Arabidopsis and rice Plant Physiol 1341718-1732
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Bastakis E Schwechheimer C (2013) Cross-repressive interactions between SOC1 and the GATAs GNC and GNLCGA1in the control of greening cold tolerance and flowering time in Arabidopsis Plant Physiol 162 1992-2004
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Muller IK Schwechheimer C (2010) The GATA-type transcription factors GNC and GNLCGA1 repressgibberellin signaling downstream from DELLA proteins and PHYTOCHROME-INTERACTING FACTORS Genes Dev 24 2093-2104
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Richter R Behringer C Zourelidou M Schwechheimer C (2013) Convergence of auxin and gibberellin signaling on the regulationof the GATA transcription factors GNC and GNL in Arabidopsis thaliana Proc Natl Acad Sci U S A 110 13192-13197 wwwplantphysiolorgon October 10 2018 - Published by Downloaded from
Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Su W Howell SH (1995) The effects of cytokinin and light on hypocotyl elongation in Arabidopsis seedlings are independent andadditive Plant Physiol 108 1423-1430
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tang H Woodhouse MR Cheng F Schnable JC Pedersen BS Conant G Wang X Freeling M Pires JC (2012) Altered patterns offractionation and exon deletions in Brassica rapa support a two-step model of paleohexaploidy Genetics 190 1563-1574
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang X Wang H Wang J Sun R Wu J Liu S Bai Y Mun JH Bancroft I Cheng F Huang S Li X Hua W Wang J Wang X FreelingM Pires JC Paterson AH Chalhoub B Wang B Hayward A Sharpe AG Park BS Weisshaar B Liu B Li B Liu B Tong C Song CDuran C Peng C Geng C Koh C Lin C Edwards D Mu D Shen D Soumpourou E Li F Fraser F Conant G Lassalle G King GJBonnema G Tang H Wang H Belcram H Zhou H Hirakawa H Abe H Guo H Wang H Jin H Parkin IA Batley J Kim JS Just J Li JXu J Deng J Kim JA Li J Yu J Meng J Wang J Min J Poulain J Wang J Hatakeyama K Wu K Wang L Fang L Trick M LinksMG Zhao M Jin M Ramchiary N Drou N Berkman PJ Cai Q Huang Q Li R Tabata S Cheng S Zhang S Zhang S Huang S SatoS Sun S Kwon SJ Choi SR Lee TH Fan W Zhao X Tan X Xu X Wang Y Qiu Y Yin Y Li Y Du Y Liao Y Lim Y Narusaka Y WangY Wang Z Li Z Wang Z Xiong Z Zhang Z Brassica rapa Genome Sequencing Project C (2011) The genome of the mesopolyploidcrop species Brassica rapa Nat Genet 43 1035-1039
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Weaver LM Amasino RM (2001) Senescence is induced in individually darkened Arabidopsis leaves but inhibited in wholedarkened plants Plant Physiol 127 876-886
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whipple CJ Hall DH DeBlasio S Taguchi-Shiobara F Schmidt RJ Jackson DP (2010) A conserved mechanism of bractsuppression in the grass family Plant Cell 22 565-578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhang X Zhou Y Ding L Wu Z Liu R Meyerowitz EM (2013) Transcription repressor HANABA TARANU controls flowerdevelopment by integrating the actions of multiple hormones floral organ specification genes and GATA3 family genes inArabidopsis Plant Cell 25 83-101
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhao Y Medrano L Ohashi K Fletcher JC Yu H Sakai H Meyerowitz EM (2004) HANABA TARANU is a GATA transcription factorthat regulates shoot apical meristem and flower development in Arabidopsis Plant Cell 16 2586-2600
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
ACKNOWLEDGMENTS
CS would like to thank Eilon Shani and Daniel Chamovitz Department of Molecular Biology and Ecology of Plants for hostingCS at Tel Aviv University during manuscript preparation QLR EB and CS would like to thank Jutta Elgner for her excellenttechnical support
wwwplantphysiolorgon October 10 2018 - Published by Downloaded from Copyright copy 2016 American Society of Plant Biologists All rights reserved