Supplemental Dat a. Wohlbach et al. (2008). Analysis of ......Apr 18, 2008 · At2g34870 hydroxyproline-rich glycoprotein family protein 0 0.026201 0.004742 -0.22 -0.45 5.35 5.57

Supplemental Data. Wohlbach et al. (2008). Analysis of the Arabidopsis histidine kinase

ATHK1 reveals a connection between vegetative osmotic stress sensing and seed maturation.

Supplemental Figure 1. Locations of athk1 T-DNA inserts and expression levels of ATHK1

RNA.

(A) Locations of athk1 T-DNA inserts. The athk1-3 allele (a3) carries a T-DNA insertion in the sixth intron, 2465

bp downstream of the ATHK1 start. The athk1-4 allele (a4) carries a T-DNA insertion in the second exon, 593

bp downstream of the ATHK1 start. For PCR of ATHK1 genomic DNA for molecular complementation,

primers 1 and 2 were used to amplify a 6 kb fragment, and primers 3 and 4 were used to amplify an 8 kb

fragment.

(B) Expression levels of wild-type (W; dark grey), athk1-3 (a3; light grey), athk1-4 (a4; light grey), athk1/ATHK1

rescued (R; dark grey), and 35S:ATHK1 overexpressor (OE; black) were measured in seeds and seedlings.

RNA extracted from three-day old seedlings was used for standard RT-PCR and quantitative RT-PCR. RNA

extracted from mature seeds was used for quantitative RT-PCR.

(C) Expression levels of wild-type (Ws) and the three 35S:ATHK1 overexpressor mutants examined in this study.

RNA extracted from three-day old seedling was used for standard RT-PCR.

(D) Expression levels of ten-day old wild-type seedlings exposed to water ± 100 mM NaCl, 1 !M abscisic acid

(ABA), 1 !M cytokinin (6-BAP), or 1 !M gibberellin (GA) (closed squares) for 24 hours. Samples of whole-

seedling tissue were collected at 0 h, 1.5 h, 3 h, 6 h, 12 h, 18 h, and 24 h. The control (0 h) levels of ATHK1

are shown with a closed circle. Values represent the relative mean expression level from four PCR reactions.

Error bars represent the SE.

Supplemental Figure 2. Altered ABA sensitivities in root elongation of athk1 alleles. Seeds

from matched lots were germinated on MS media for three days. Seedlings were then transferred to MS media ±

ABA as indicated. Photographs of representative roots were taken five days after transfer to ABA media.

Supplemental Figure 3. athk1 knockout mutants display normal embryo development. In

order to harvest seeds of a defined developmental stage, flowers of athk1 ((A) – (D)) and wild-type (Ws, (E) – (H))

were tagged on the day of flowering with colored tape. Siliques were collected on the same day and were viewed

using bright field microscopy.

(A) The heart stage athk1 embryo at 6 DAF.

(B) The torpedo stage athk1 embryo at 7 DAF.

(C) The upturned-U stage athk1 embryo at 8 DAF.

(D) The mature cotyledon stage athk1 embryo at 10 DAF.

(E) The heart stage wild-type embryo at 6 DAF.

(F) The torpedo stage wild-type embryo at 7 DAF.

(G) The upturned-U stage wild-type embryo at 8 DAF.

(H) The mature cotyledon stage wild-type embryo at 10 DAF.

Supplemental Figure 4. Gene Ontology (GO) annotations for genes significantly

differentially regulated according to both ATHK1 transcript level and sorbitol stress

condition. Genes were classified as significantly differentially regulated if the Benjamini and Hochberg corrected

p-values for a two-way ANOVA test between log2 RMA-process expression values was less than 0.01.

Significantly enriched GO annotations are indicated with an asterisk (*).

Supplemental Figure 5. qRT-PCR measures of expression confirm microarray expression

changes. Nine genes were selected from the list of significantly differentially expressed genes generated from

microarray experiments. For microarray expression, data from the three biological replicate microarray samples

were preprocessed according to the RMA algorithm, the average expression level for each gene was calculated, and

the value was then actin-normalized. The range of variation in expression levels for the microarray replicates of

these nine genes was on average 11.3% ± 2.6%. RNA from the first biological replicate microarray samples was

used for the qRT-PCR experiments. For qRT-PCR, four technical replicate samples for each condition were

processed using the Miner algorithm to account for differences in PCR efficiencies of different primer pairs, and an

actin-normalized average expression level was calculated for each gene. The range of variation in expression levels

for the qRT-PCR replicates of these nine genes was on average 3.7% ± 3.2%. Linear fold changes

were computed for each comparison and are shown on these graphs. The calculated values are shown above or

below each corresponding column. Note that a linear fold change of 1 actually indicates no difference in average

expression values between two experimental conditions.

(A) Comparison of wild-type sorbitol stress expression (Ws sorbitol vs. Ws).

(B) Comparison of mutant profile expression (athk1 vs. Ws and 35S:ATHK1 [OEX] vs. Ws).

(C) Comparison of mutant profile expression during sorbitol stress (athk1 sorbitol vs. Ws sorbitol and 35S:ATHK1

[OEX] sorbitol vs. Ws sorbitol).

Supplemental Figure 6. Type-A ARR mutants display altered osmotic stress germination

response. Wild-type (Col-0) (diamonds), arr3,4,5,6 (circles), arr5,6,8,9 (triangles), and arr3,4,5,6,8,9 (squares)

from matched seed lots were scored for germination on the indicated concentrations of sorbitol. The percent

germination after five days of stress treatment is shown. Each value represents the mean percent germination for at

least four replicates of at least 50 seeds. Error bars represent SE.

Supplemental Figure 7. Functional distribution of gene clusters from the AtMegaCluster.

The 22,810 genes represented on the Affymetrix ATH1 microarray were hierarchically clustered and highly similar

genes were grouped into five clusters, shown in Figure 4: A – “Unclassified Without Homolog”; B –

“Environmental Information Processing”; C – “Metabolism”; D – “Cellular Processes”; E – “Genetic Information

Processing”. The genes in each cluster were functionally classified using the GeneBins database . Functional

classifications significantly enriched in a cluster over that of the whole genome (p < 0.01, with Bonferroni

correction) are represented with an asterisk (*).

Supplemental Table 1. Genes significantly differentially regulated according to ATHK1

transcript level.

log2 FC log2 Expression

Locus ID Annotation FunCata

1F-ANOVA

p-value

T-Test

p-valueb

ath

k1

/ W

s

35

S:A

TH

K1

/ W

s

ath

k1

Ws

35

S:A

TH

K1

At2g17820 ATHK1 histidine kinase 1 1,3,6,7 8.79E-09 0.000760 -3.90 2.76 3.62 7.52 10.28

athk1 / Ws Significant

At5g13330 RAP2.6L ERF subfamily B-4 of ERF/AP2 transcription factor 2,4 0.027944 0.007595 -0.32 0.20 6.97 7.29 7.49

At4g36410 UBC17 ubiquitin-conjugating enzyme 17 3,4 0.039816 0.009473 -0.30 0.10 7.20 7.50 7.61

At2g34870 hydroxyproline-rich glycoprotein family protein 0 0.026201 0.004742 -0.22 -0.45 5.35 5.57 5.12

At2g17920 expressed protein, similar to zinc finger protein 0 0.049546 0.005664 -0.09 -0.16 3.65 3.74 3.59

At4g32170 CYP96A2 cytochrome P450 4,5 0.001843 0.007464 0.04 -0.08 3.83 3.79 3.71

At1g28710 expressed protein 0 0.001992 0.007488 0.14 -0.07 5.09 4.95 4.88

At3g24515 UBC37 ubiquitin-conjugating enzyme, putative 3,4 0.021735 0.009823 0.20 -0.08 5.35 5.16 5.08



At1g42605 unknown 0 0.048739 0.009895 0.37 -0.07 5.28 4.91 4.84

At1g48070 expressed protein 0 0.027669 0.002053 0.42 0.46 5.00 4.58 5.03

35S:ATHK1 / Ws Significant

At4g25680 expressed protein 0 0.022229 0.008816 -0.07 0.95 8.08 8.15 9.10

At2g02140 LCR72 plant defensin-fusion protein, putative 7 0.022496 0.009701 0.01 0.57 4.74 4.73 5.30

At2g31380 STH zinc finger (B-box type) / salt tolerance-like protein 2,4 0.000725 0.003813 0.12 0.56 6.55 6.42 6.99

At3g54140 proton-dependent oligopeptide transport 5 0.001716 0.005082 -0.19 0.55 9.45 9.64 10.20

At1g27520 glycoside hydrolase family 47 protein 1,3 0.000546 0.003583 -0.03 0.52 4.09 4.12 4.64

At2g26920 ubiquitin-associated (UBA)/TS-N domain-containing 0 0.004391 0.006556 -0.04 0.47 4.46 4.50 4.97

At1g04880 high mobility group / ARID/BRIGHT DNA-binding domain 2 0.046060 0.010061 0.04 0.42 7.05 7.01 7.43

At2g21490 dehydrin family protein 7 0.006012 0.007039 0.02 0.39 7.29 7.28 7.66


At3g02590 delta 7-sterol-C5-desaturase, putative 1 0.002507 0.005732 0.18 0.37 6.50 6.33 6.70


At1g29720 protein kinase family protein 1,3 0.000002 0.001427 0.11 0.34 4.98 4.87 5.21





At5g44090 calcium-binding EF hand family protein, putative 4 0.004345 0.006266 0.10 0.24 4.00 3.90 4.13

At3g56860 UBA2A UBP1 interacting protein 2a 2,4,6 0.011712 0.007480 0.05 0.23 5.19 5.13 5.37

At5g50780 ATP-binding region, ATPase-like domain-containing 4 0.000876 0.004538 0.04 0.23 7.75 7.71 7.94

At5g19290 esterase/lipase/thioesterase family protein 0 0.001163 0.004710 0.05 0.22 8.45 8.41 8.63


At4g35985 senescence/dehydration-associated protein-related 0 0.043299 0.009921 -0.03 -0.17 4.15 4.18 4.01

At1g77250 PHD finger family protein 2,4 0.005939 0.006707 0.00 -0.18 5.72 5.72 5.54

At5g64980 expressed protein 0 0.013246 0.008152 -0.02 -0.21 4.57 4.59 4.38

At3g55320 PGP20 ABC transporter family protein 1,4,5 0.003216 0.006072 0.02 -0.24 10.18 10.16 9.92



At2g46830 CCA1 myb-related transcription factor 2,7 0.008727 0.007181 -0.06 -0.29 4.21 4.26 3.97

At5g08370 similar to alpha-galactosidase, putative / melibiase, putative 1 0.000251 0.002354 0.03 -0.29 5.45 5.42 5.12

At5g52190 sugar isomerase (SIS) domain-containing protein 1,4 0.000593 0.003756 0.02 -0.30 4.88 4.85 4.55

At3g43690 unknown 0 0.023687 0.009749 0.01 -0.33 5.63 5.62 5.29

At1g43330 myb family protein-related 2 0.017679 0.008409 -0.26 -0.34 4.02 4.29 3.95

At1g10320 U2 snRNP auxiliary factor-related 4 1.77E-07 0.001376 -0.21 -0.49 5.99 6.20 5.71

At1g05900 endonuclease-related 1,2,4 1.72E-07 0.000499 0.06 -0.57 5.56 5.51 4.93

Only ATHK1 was significantly differentially regulated in both athk1 and 35S:ATHK1 mutant backgrounds. Other

genes significantly differentially expressed in either athk1 or 35S:ATHK1 are shown here.

a Genes were grouped into functional categories using the MIPS FunCatDB : 1 – metabolism; 2 – transcription; 3 –

post-translation (folding, modification, destination); 4 – binding; 5 – transport; 6 – signal transduction; 7 – abiotic

stress response; and 0 – unclassified/other.

b p-values from an unpaired t-test between athk1 and Ws control or 35S:ATHK1 and Ws control signal intensities

were corrected using the method of Benjamini and Hochberg. A significance level of " = 0.01 was used as the cut-

off.

Supplemental Table 2. Common experimental conditions in the ATHK1 gene cluster.

Experimenta

ATHK1 ARR5 ARR9 ARR6 ARR7 ARR4

unknown

protein

(At4g37080) ARR8

18S pre-

ribosomal

assembly

protein

(At1g13650)

Abiotic Stress

6h Heat (root) -2.43 -1.73

24h Mannitol (root) -2.36 -1.89 -1.23 -1.26

6h Mannitol (root) -2.26 -1.70

12h Mannitol (root) -2.09 -1.74 -1.38 -1.26

24h Salt (root) -1.73 1.51

6h Salt (root) -1.73 -1.96 1.45 -1.91 -1.50

12h Salt (root) -1.71 -1.72 -1.25

1h Drought (root) -1.49 -1.66 -1.63 -2.09 -1.16

6h Heat (root) -1.43 -1.91

24h Mannitol

(shoot)

-1.26 -2.20 -1.68 -2.87 -2.78 -2.31 -2.27 -3.59

Other

ARR22-OEX 3h

1!M t-Zeatin

-1.42 -5.00 -3.38 -4.36 -5.70 -5.19 -1.31 -2.69

3h 10!M

Cycloheximide

-1.53 2.32 -1.38 3.32 -1.28

24h 30!M ABA

(seed)

1.07 -1.39 1.36 1.03

3h 1!M t-Zeatin 1.16 2.81 1.62 1.91 1.28 1.48

3h 20!M t-Zeatin 1.22 2.48 1.16 2.45 2.51 1.97 1.55

2h P. syringae

DC3000

1.47 2.16 -3.29 -1.36 3.66

1h 1!M t-Zeatin 1.48 2.75 1.32 2.06 1.26 1.61

96h H2O (seed) 1.67 -1.64 -1.40 -1.81

ARR21c-OEX 2.27 3.97 1.36

A. tumefaciens

tumour induction

3.56 2.03 2.69 2.23 1.39

a Experiments in which the log2 fold change of ATHK1 and at least one other gene in the ATHK1 cluster is greater

than 1.0 (for induced genes) or less than -1.0 (for repressed genes) are shown here. For simplicity, expression values

that do not pass this fold change cut-off are not shown. Bold text indicates that all fold changes passing our cut-off

are in the same direction.

Supplemental Table 3. Sequences of primers used for qRT-PCR analysis.

Name Sequence Tm (˚C) %GC Length

cDNA

Product

gDNA

Product

FUS3-RTF1 GGGTTATCGGCGTCTGTGCCTCT 63.2 58.3 24

FUS3-RTR1 TCCGCGGCTTTCTTCGGGAGTA 63.6 59.1 22 252 524

LEC1-RTF1 CGGCGCCGGTGACAAGAAC 60.9 68.4 19

LEC1-RTR1 GACGAAGAGCCACCACCAACACTG 61.6 58.3 24 555 555

CRA1-RTF1 AGCGCCAGATGCACCGATAAC 60.8 61.9 21

CRA1-RTR1 GTTCGCGTTTGCGTTCCACTG 60.1 57.1 21 186 301

CRU2-RTF1 TCTCCGCCTTAGCGCTCTTCGTG 63.5 60.9 23

CRU2-RTR1 CCTGTGCGTTTTCGTTTGTCTTGA 60.0 45.8 24 263 504

CRU3-RTF1 CGACATCGCCAACTACCAAAACCA 61.6 50.0 24

CRU3-RTR1 CGTCGAACCCGCTCCACAAGT 60.8 61.9 21 134 259

At2S1-RTF1 ATCTACCGCACCGTCGTTGAGTTC 60.2 54.2 24

At2S1-RTR1 GCCTTGCCTTGCTTGCTGGAG 60.8 61.9 21 144 144

At2S2-RTF1 TGGCAAACAAGCTCTTCCTCGTCT 60.2 50.0 24

At2S2-RTR1 GGGTTGCTGGCGTCATCTTCGTC 63.5 60.9 23 113 113

At2S4-RTF1 CGCAAGCAAATGTGGCAAGGAC 61.3 54.5 22

At2S4-RTR1 AGGTGGGGCAAACGCAAACTG 60.7 57.1 21 154 154

RAB18-RTF1 AGGTGGCCAAGGATACGGAACAGG 63.0 58.3 24

RAB18-RTR1 GCCCATCGCTTGAGCTTGACCAG 63.7 60.9 23 321 402

LEA14-RTF1 GGCCAAAGTCTCTGTCACCAATCCT 60.5 52.0 25

LEA14-RTR1 GCCGTCATGTCCTTAGCTTTCAGAGA 60.9 50.0 26 141 217

RD20-RTF1 TACGGAACGATTTGGAGGAAACATTACC 62.1 42.9 28

RD20-RTR1 CACTCATTCCTTTGCTATCGTGACCTTCTG 63.8 46.7 30 114 388

RD29B-RTF1 GAGGAGGATCGGATTATCTCAGTGGTGTAT 61.8 46.7 30

RD29B-RTR1 AGTCTTCTTCGCGTCCTTGTCTTGATTTCT 63.6 43.3 30 171 241

ABI2-RTF1 ACTTGAGTGCTCATTTCTTTGGTGTTTACG 61.8 40.0 30

ABI2-RTR1 GTTTCTCCTTCACTATCTCCTCCGTCAAAG 61.8 46.7 30 114 196

CCA1-RTF1 ATAACAAAGCAACGTGAAAGGTGGACTGAG 63.4 43.3 30

CCA1-RTR1 AATTTCTGAGCGTGACTTCTTATCTGGACA 61.6 40.0 30 146 235

ATMYB2-RTF1 GTTAAACAACGCAACTTCTCAAAAGATGA 59.2 34.5 29

ATMYB2-RTR1 GTTTCCCAATGGATCCTAACTTCGTT 58.8 42.3 26 137 223

ATHB12-RTF1 CCGAGGCAAGTGGCTATATGGTTTCAG 63.5 51.9 27

ATHB12-RTR1 TGTGTCTTCTTTTGCGTCGCCTCTTTTA 63.8 42.9 28 191 411

KIN1-RTF1 ACAAGAATGCCTTCCAAGCCGGTCAGAC 67.5 53.6 28

KIN1-RTR1 CGATACACTCTTTCCCGCCTGTTGTGCT 66.6 53.6 28 131 412

Primers are listed in the 5’ to 3’ direction. Primers were designed around introns to have a cDNA product

approximately 100 to 300 bp long. Primer melting temperatures were calculated using the program PrimerSelect in

the DNAStar Lasergene software package (http://www.dnastar.com). Optimal annealing temperatures for each

primer pair were determined experimentally.

Supplemental Table 4. Replicate quality of biological duplicate microarray experiments.

Genotype Replicate Comparison r2 Value

a

chip 1 vs. chip 2 0.9847

chip 1 vs. chip 3 0.9708 WT



chip 1 vs. chip 3 0.9596 WT + sorbitol



chip 1 vs. chip 3 0.9856 athk1



chip 1 vs. chip 3 0.9855 athk1 + sorbitol



chip 1 vs. chip 3 0.9784 Rescue



chip 1 vs. chip 3 0.9851 Rescue + sorbitol



chip 1 vs. chip 3 0.9882 35S:ATHK1



chip 1 vs. chip 3 0.9824 35S:ATHK1 + sorbitol


a The r2 linear regression value was computed for all pairs of array replicates. A value of 1 means perfect

correlation for gene expression measures. Except for comparisons to Rescue chip 2, the correlation between

replicates was never less than 0.95.

Documents

Supplemental Dat a. Wohlbach et al. (2008). Analysis of ......Apr 18, 2008 · At2g34870 hydroxyproline-rich glycoprotein family protein 0 0.026201 0.004742 -0.22 -0.45 5.35 5.57