Supplementary Materials for - Science SignalingSupplementary Materials for Combinatorial interaction network of transcriptomic and phenotypic responses to nitrogen and hormones in

Supplementary Materials for

Combinatorial interaction network of transcriptomic and phenotypic

responses to nitrogen and hormones in the Arabidopsis thaliana root

Daniela Ristova, Clément Carré, Marjorie Pervent, Anna Medici, Grace Jaeyoon Kim,

Domenica Scalia, Sandrine Ruffel, Kenneth D. Birnbaum, Benoît Lacombe,

Wolfgang Busch, Gloria M. Coruzzi, Gabriel Krouk*

*Corresponding author. Email: [email protected]

Published 25 October 2016, Sci. Signal. 9, rs13 (2016)

DOI: 10.1126/scisignal.aaf2768

The PDF file includes:

Fig. S1. Hormones and nitrogen interact to shape root system architecture

(absolute quantification).

Fig. S2. Revealing genome-wide features of nitrogen-hormone interactions, the

top 100 models.

Fig. S3. Study of the NO3*CK-only responsive genes.

Fig. S4. qRT-PCR validation of sentinel genes.

Fig. S5. Sungear figures (generalized Venn diagram) measuring the convergence

of nutritional and hormonal signaling pathways.

Fig. S6. Identification of marker genes of simple signals.

Fig. S7. Multivariate model with signal-to-trait connections.

Fig. S8. Clustering analysis of genes correlated with root traits.

Fig. S9. Clustering of all the genes controlled by NO3−.

Fig. S10. Clustering of all the genes controlled by NH4+.

Fig. S11. Clustering of all the genes controlled by IAA.

Fig. S12. Clustering of all the genes controlled by CK.

Fig. S13. Clustering of all the genes controlled by ABA.

Fig. S14. Characterization of mutants at the transcriptional level.

Fig. S15. Mutation in a K+ channel affects root development and ABA

responsiveness on nitrate-containing media.

Fig. S16. Venn diagram illustrating the influence of signal combinations on the

whole root transcriptome.

Table S1. Matrix of treatments applied in this study.

www.sciencesignaling.org/cgi/content/full/9/451/rs13/DC1

Legends for tables S2 and S3

Table S4. Best marker genes for the simple signals.

Other Supplementary Material for this manuscript includes the following:

(available at www.sciencemagsignaling.org/cgi/content/full/9/451/rs13/DC1)

Table S2 (Microsoft Excel format). Genome-wide ANOVA results.

Table S3 (Microsoft Excel format). GeneCloud analysis of the 10 dominant

modes of regulation and gene clusters.

Data File S1. Nitrogen+Hormones.cys Cytoscape file for Fig. 3B.

Figure S1. Hormones and nitrogen interact to shape root system architecture (absolute

quantification). Box plots (n = 12–20 plants measured in at least 2 independent experiments) of

the different root parameters in response to an exhaustive combination of 5 signaling molecules

(nitrate, ammonium, auxin, cytokinin, and ABA). P: Primary root length; P1: Primary root length

before treatment; P2: primary root growth after treatment; LR: lateral root; LR.nb: total number

of visible (>0.5 mm) lateral roots; LR.nb.P1: number of visible lateral roots in P1; LR.length:

Mean of lateral roots; LR.dens: number of lateral roots per cm of primary root; LR.P.ratio: ratio

between LR length and P length; T.LR.L: Total LR length; T.R.L: Total root length (P plus LR

length).

Number'of'probes'

3499'

Mod

el'#'

Figure S2. Revealing genome-wide features of nitrogen-hormone interactions, the top 100 models.Dominant models of gene regulation classified by the number of probes affected by a given modelof regulation.

Figure S3. Study of the NO3*CK-only responsive genes. (A) Heat map of the 328 genes having NO3*CK as the only significant term controlling them (see Fig. 2A). (B) GeneCloud results highlighting the most highly represented semantic terms in this gene list controlled by the NO3*CK composite signal alone. (C) GeneCloud table of the most highly represented semantic term sorted by p-value.

Figure S4. qRT-PCR validation of sentinel genes. qRT-PCR validation of sentinel genes reporting hormone [retrieved from ref Nemhauser et al, 2006] and nitrogen treatments. Data are means ± SEM, n = 6 from 3 independent experiments performed on different days.

NO3-

NH4+

IAA

CK

ABA

mR

NA

leve

l

NAC4

ARR8

IAA19

NIR1

NRT2.1

Figure S5. Sungear figures (generalized Venn diagram) measuring the convergence of

nutritional and hormonal signaling pathways. Each vessel represents a set of genes genes that

are similarly regulated pointing to vertices (the simple or composite signals indicated). The size

of the vessel is proportional to the size of the corresponding gene set (64, 65).

Figure S6. Identification of marker genes of simple signals. Pattern matching was used to

uncover genes that were mainly regulated by a simple signal. Pattern matching consisted of

correlating (Pearson) gene expression to an idealized model of regulation corresponding to each

signal. Because each signal has a different strength (Fig. 2), the threshold used has been

incrementally modified (by 0.5 steps) in order to retrieve more than 5 genes and have an overall

correlation with the idealized model >0.6. The thresholds used here are: Nitrogen (>0.65), NO3-

(>0.7), NH4+ (>0.6), CK (>0.75), IAA (>0.8), ABA (>0.9).

NO3-

NH4+

IAA

CK

ABA

NO3-

NH4+

IAA

CK

ABA

Nitrogen

Figure S7. Multivariate model with signal-to-trait connections. This is a modified version of

the multivariate network model presented Fig. 2B, where signals are connected to traits according

to ANOVA results. The network was then displayed using the Cytoscape layout function named

‘force directed,’ which displays nodes according to their connectivity (Force of the edges are un-

weighted, all even). This displays clusters of nodes (including genes) that are related to the

signals and to the traits. These clusters are subjected to GeneSect in order to identify biological

functions that are over-represented in each cluster.

interpro−ipr006459(4/60)interpro−ipr006702(4/40)

upf0497(4/40)tran−membrane(4/36)

previously(2/41)radiation(2/39)

abundant(5/15)substrate(7/7)

biosynthesis(7/6)

interpro−ipr015467(2/21)

sugar−hydrogen(3/14)interpro−ipr002067(2/20)

late(4/10)lea(3/13)


rate(2/18)

uncharacterised(4/9)

nucleolus(4/8)


sugar−inositol(2/15)

form(4/7)


phosphate(4/7)

senescence(3/9)



substrate−solute(2/13)

biogenesis(4/6)

symporter(3/8)

water(3/8)



phospholipase(2/11)

bifunctional(3/7)

embryogenesis(3/7)


major(5/4)

endonuclease(2/9)

oxidative(3/6)

ribonucleoprotein(2/9)

subdomain(3/6)

well(2/9)

inter pro−ipr015421(2/8)

inter pro−ipr015424(2/8)

At4g14890

At5g66170

At4g15680

At3g43800

At1g71880

At5g28770

At4g07820

At5g54840

At1g68795

At5g63600

At3g26090

At3g21520

At4g21850At5g03310

At5g14530

At3g62280At2g30550

At5g37500

At1g05450

At3g30210

At5g23020

At3g14940

At5g59540

At5g13520

At2g40170

At5g25640

At3g06700

At2g01580

At1g03210

At4g11290

At3g14560

ABA

At5g27350

At4g15290

At4g15690

At2g22170

At4g04830

At5g63790

Prim

At3g01260At2g47540

At4g34710

Total.LR

At3g10640LR.Length.dens

At3g07070

At1g76090

At1g70990

At3g56880

At3g10720

At4g19030

At4g30170

At1g53680

At4g37295

At4g19980

At1g02570

At3g54400

At1g05260

At5g07580

At4g01140

At3g56230

At4g21200

At3g58000

At2g39705

NH4

LR.density

AND 3

At5g02420

NO3

LR.lenghtAND 2

At3g44540

At2g30860

At4g04840

At3g01190

At4g25260

At2g39530

At4g17350

At3g59900

At3g21680

At1g22440

P2

AND 4

At4g15770

At3g16690

Total.Root

At5g56540

At3g58190

At5g25240

At5g18060

At2g39370

At3g02000

At4g11280

At5g18560

At1g75750

At1g24620

At5g55200

At5g47440At5g23710

At5g26930

At4g14690

At2g25810

At5g10430

At4g32280

At2g16980

At1g62510

At1g75780

At1g72160

At3g59370

At4g08950

At1g12090

At2g25790

At4g22460

At1g73490

At5g19530

At3g05490

At2g33310

At3g23805

At1g78100

At5g45280

At1g51420

At5g47370

At1g29500

At5g67280

At3g45110

At5g57340

At1g13910

At5g60660At3g63470

At3g63440

At3g26760At4g27590

At3g22570

At3g20830

At4g14640

At5g62310

At5g06860

At3g09280

At4g21730

At1g16390

At1g63420

At3g15540

At1g80240

At4g26320

At3g60420

At3g06460

At2g14960

At1g73780

At1g19900

At3g21340

At3g13720

At5g16190

At2g31390

At3g06730

At5g59870

At5g23460

At1g67060

At5g61440

At3g44380At3g19040

At1g11190

At4g23493

At1g52200

At1g61930

At1g10840

At2g28790

At5g53120At4g21310

At3g47420

At3g45160

At3g27200

At2g44740At5g20160At5g57610

At1g03700

At1g54890

At2g35760

At1g78610

At2g37870

At1g25580

At3g30350

At5g59090

At3g09085

At3g58810

At4g15610

At2g17300

At2g43390

At4g33660

At3g13730

At4g11570

At2g02590

At5g19410

At5g01820

At1g04870

At5g01500

At3g11780

At3g62260

At1g52930

At3g06750

At5g61810

At3g48730

At4g21980

At4g22212

At5g20850At4g15630

At2g40110

At1g28400

At1g50170

At2g22850

At3g56730

At1g07680

At3g05150

At1g73500

At2g05530

At3g01120

At4g14270

At5g52470

At3g19930

At4g32551

At4g33790

At4g13195

At3g44450

At2g17680

At1g08500

At5g44460

At1g50720

At3g54770

At4g04900

At3g51660

At4g40090

At1g01750

At2g41100

At5g55650

At4g03320

At1g14220

At4g23070

At1g43910

At1g03870

At2g02620

At5g16590

At5g44020

At4g31320 At3g12760

At2g34140

At1g02850

At2g43550

At3g23030

At5g47950At4g13440

At1g07300

At5g53250

At1g72430

At1g51830

At2g23170

At1g76930

At2g45420

At2g34650

At5g17340

At3g54960

At3g43960

At4g37470

At2g45180At4g23400

At3g48970

At5g57520

At4g09890

At2g39040

At4g12080 At1g77640

At5g54490 At1g23205

IAA

CK

At2g31085

LR.nb

LR.nb.P1 At1g05680

At5g56870

At2g41730

LR.dens.P1

longer(3/68)cgmp−dependent(2/75)

camp−dependent(2/66)

agc−kinase(2/62)



storage−trypsin−alpha(5/24)amylase(5/23)

auxin−responsive(5/22)

inhibitor−lipid−transfer(5/22)


protein−hydrophobic(4/27)

albumin(5/21)

inhibitor−plant(5/21)interpro−ipr016140(5/21)

auxin(8/13)

root(17/6)

aux−iaa−arf−dimerisation(3/33)interpro−ipr003311(3/33)


protein−seed(5/19)

aux−iaa(3/30) transfer(6/15)

downregulated(2/40)

arabinogalactan(3/25)

bifunctional(5/15)


storage(5/14)

aquaporin(2/33)


strong(2/33)

agc(2/32)

hair(4/16)

diversely(2/31)


ralf−like(2/29)


stimulus(7/8)

alkalinization(2/26)

ralf(2/26)

showing(2/26)

physiology(2/24)

transport(12/4)

exhibit(3/14)

inhibitor(6/7)

rapid(2/21)

cyclopentenone(2/20)

believed(2/18)

boundary(2/18)

inducible(2/18)

unidimensional(2/18)


characterized(2/17)

brassinosteroid(2/15)

helical(5/6)

lateral(3/10)

lipid(5/6)

red(2/15)

consist(2/13)


saur(2/13)

saur−like(2/13)

tobacco(2/13)

major(5/5)anthesis(8/3)

calcium−binding(3/8)

factor(12/2)



morphogenesis(2/12)

exon(2/11)

vivo(2/11)

specific(3/7)


leucine−rich(5/4)

n−terminal(10/2)

signal(5/4)

elongation(3/6)

localization(2/9)

peptide(3/6)

play(3/6)

whor l(3/6)

integral(4/4)

interpro− ipr002048(2/8)

signaling(4/4)

induced(3/5)

ef−hand−like(2/7)

inter pro− ipr011992(2/7)


result(2/7)

single(2/7)

water(2/7)


inhibitory(2/116)1−4(2/78)

arabinogalactan(3/34)


motif−containing(3/27)

egg(2/36)

root(11/6)

temperature(2/25)anchored(3/15)

calmodulin(3/15)seedling(4/10)

belonging(2/17)


shoot(4/8)

inhibitor(4/6)

invertase−pectin(2/12)


vascular(2/11)

anthesis(7/3)

endopeptidase(3/7)

methylesterase(2/10)

apex(2/9)ef−hand−like(2/9)

endoplasmic(3/6)




pectinesterase(2/9)

plant−type(3/6)

reticulum(3/6)

ef−hand(2/8)

six(2/8)

expanded(3/5)

storage(2/7)

pid−binding(2/1598)pbp1(2/1065)

pid(2/639)pinoid(2/639)

auxin(5/25)

act(3/15)single(2/20)

ef−hand(2/18)


calcium−binding(2/16)interpro−ipr017853(2/15)

signaling(3/9)

endomembrane(7/3)

anthesis(4/5)

compound(2/10)induced(2/10)

root(4/5)

acid(6/3)

containing(3/6)

motif(3/6)

differentiation(4/4)

expansion(4/4)

petal(4/4)putative(4/4)

dna−binding(3/5) flower(3/5)

phosphorylation(3/5)

sequence−specific(3/5)

glycoside(2/7)

inhibitor(2/7)

carbohydrate(2/6)

dna(4/3)

may(2/6)

peroxidase(3/86)interpro−ipr019794(2/102)

heam−ligand(2/86)interpro−ipr019793(2/86)

interpro−ipr000823(2/82)interpro−ipr002016(2/73)

plant−fungal−bacterial(2/73)interpro−ipr010255(2/72)

haem(2/62)

oxidative(2/31)

root(5/11)

oxidation(3/14)heme(2/20)hypocotyl(2/19)

reduction(3/12)stress(2/8)

transferase(2/6)

g−protein(2/34)interpro−ipr012335(2/23)


thioredoxin(2/20)

thioredoxin−like(2/20)

gtpase(2/17)

reductase(2/15)

electron(2/11)

carrier(2/10)fold(3/6)

root(3/5)

subgroup(2/5)

alcohol(2/34)hair(2/28)

reductase(3/17)

root(6/8)

reduced(2/15)

Figure S8. Clustering analysis of genes correlated with root traits. (A) Clustering analysis of

genes correlated with root traits in our data set in the Vanneste et al. (2005) dataset. Genes

correlated with lateral root initiation (LRI) are marked by a red spot in the LRI column. This

overlap is larger than expected by chance based on a randomization test (Genesect). (B)

Clustering analysis of genes correlated with root traits in our data set in the data set from Lavenus

et al. (2015), who analyzed gene expression dynamics upon gravistimulation, leading to root

initiation and development. The influence of the simple (single) signals (current study) are

represented by color coding in the columns to the right of the image (ABA in green, IAA in red,

NO3- in Cyan, CK in blue, NH4

+ in purple).

La

tera

l R

oo

t in

itia

tio

n g

en

es

ov

erl

ap

(40

) is

H

igh

er

tha

n e

xp

ecte

d (

Zs

co

re=

9)

A B

Time after gravistimulation

Figure S9. Clustering of all the genes controlled by NO3

−.

NO3- responsive probes (720)

Figure S10. Clustering of all the genes controlled by NH4+.

NH4+ responsive probes (767)

Figure S11. Clustering of all the genes controlled by IAA.

IAA responsive probes (1301)

Figure S12. Clustering of all the genes controlled by CK.

CK responsive probes (995)

Figure S13. Clustering of all the genes controlled by ABA.

ABA responsive probes (5799)

Figure S14. Characterization of mutants at the transcriptional level.standard MS/2 media, and transcript accumulation SEM, n = 3.

Characterization of mutants at the transcriptional level. Plants weand transcript accumulation was measured by qRT-PCR. Data are means ±

were grown on PCR. Data are means ±

Figure S15. Mutation in a K+ channel affects root development and ABA responsiveness on nitrate-containing media. Plants (WT and gork mutants) were grown on 0.5 mM NH4NO3 media for 10 days and then transferred to fresh media containing 0.5 mM NH4NO3, with or without 1µM ABA. The plates were photographed each day thereafter for 5 days. (A) Analysis of variance (ANOVA) results for total root length across time points (day 1 to day 5). (B) Two independent mutant gork lines (salk_082258 in the Col-0 background and gork-1 in WS background) display phenotypes in root development and in the root response to ABA treatment. Data are presented as mean +/- SEM, n=8–9. Asterisks represent the time points at which the interaction between the genotype and the ABA response are significant (p<0.05). (C) At day 5 plants were harvested and meristem size (from quiescent center to the first elongating epidermal cell) measured. Left: Lateral root meristem measurements on plants treated for 5 days with ABA as described above. Data are presented as mean +/- SEM, n=19–33. Right: Swellings of epidermal cells observed in both the primary root (Prim) and lateral root (LR) in salk_082258 after 10 days of growth in the presence of ABA. These are representative pictures taken from a high number (>20) of observations performed in different experiments in different labs.

0"

20"

40"

60"

80"

100"

120"

140"

[mm]"

Ws"no:ABA"

Ws"ABA"

gork:1"no:ABA"

gork:1"ABA"

Prob>|t| TRL_d1 TRL_d2 TRL_d3 TRL_d4 TRL_d5

Genotype[salk01] <.0001 0.2509 0.0455 0.0015 0.0038

Condition[ABA] 0.4409 0.0687 0.2698 0.1718 0.0941

Genotype[salk01]*Condition[ABA] 0.5121 0.2966 0.3025 0.0435 0.0135

Genotype[gorkF1] 0.1204 0.7701 0.2012 0.1438 0.0014

Condition[ABA] 0.2231 0.0469 <.0001 <.0001 <.0001

Genotype[gorkF1]*Condition[ABA] 0.7977 0.3654 0.0622 0.0719 0.0017

*

0"

20"

40"

60"

80"

100"

[mm]"

Col:0"no:ABA"

Col:0"ABA"

salk01"no:ABA"

salk01"ABA"

*

*

To

tal

roo

t le

ng

th

Days a er transfert

1 2 3 4 5

B

A

salk_082258 Col gork-1 WS

Prim LR 0"

50"

100"

150"

200"

250"

300"

C

Late

ral

roo

t m

eri

ste

m s

ize (

µm

)

- + - + - + - + ABA

WS

gork-1

Col

salk_082258

Col

a a

b

c

x x

x

y

Figure S16. Venn diagram illustrating the influence of signal combinations on the whole root transcriptome. Probes regulated by simple signals (ABA, IAA, CK, NO3, or NH4) or by composite signals (IAA*CK or IAA*CK*ABA... for example) have been retrieved. A Venn diagram has been generated to compare the different probes regulated by simple or composite signals.

=Controlled by at least a composite signal

e.g: CK*IAA, NO3*NH4, CK*NO3 …

Controlled by at least a simple signal =

e.g: CK, IAA, NO3, NH4 …

Treat #

KN

O3

(1

mM

)

NH

4C

l

(1

mM

)

Au

xin

(5

00

nM

)

Cyto

kin

in

(5

00

nM

)

AB

A

(1

µM

)

KC

l

(1

mM

)

DM

SO

1 0 0 0 0 0 2 3

2 0 0 0 0 1 2 2

3 0 0 0 1 0 2 2

4 0 0 0 1 1 2 1

5 0 0 1 0 0 2 2

6 0 0 1 0 1 2 1

7 0 0 1 1 0 2 1

8 0 0 1 1 1 2 0

9 0 1 0 0 0 1 3

10 0 1 0 0 1 1 2

11 0 1 0 1 0 1 2

12 0 1 0 1 1 1 1

13 0 1 1 0 0 1 2

14 0 1 1 0 1 1 1

15 0 1 1 1 0 1 1

16 0 1 1 1 1 1 0

17 1 0 0 0 0 1 3

18 1 0 0 0 1 1 2

19 1 0 0 1 0 1 2

20 1 0 0 1 1 1 1

21 1 0 1 0 0 1 2

22 1 0 1 0 1 1 1

23 1 0 1 1 0 1 1

24 1 0 1 1 1 1 0

25 0.5 0.5 0 0 0 1 3

26 0.5 0.5 0 0 1 1 2

27 0.5 0.5 0 1 0 1 2

28 0.5 0.5 0 1 1 1 1

29 0.5 0.5 1 0 0 1 2

30 0.5 0.5 1 0 1 1 1

31 0.5 0.5 1 1 0 1 1

32 0.5 0.5 1 1 1 1 0

Table S1. Matrix of treatments applied in this study. All combinations of the 5 signaling

molecules: nitrate (KNO3), ammonium (NH4Cl), auxin, cytokinin (CK), abscisic acid (ABA)

were performed, including the mock treatments (KCl and DMSO).

Table S2. Genome-wide ANOVA results. The table, provided in a separate Excel file

(ANOVA_Results.xlsx), contains p-values for each probe for their response to simple and

composite signals.

Table S3. GeneCloud analysis of the 10 dominant modes of regulation and gene clusters. This table is provided in a separate Excel file (GeneCloud_Results.xlsx). 14 sets of genes

controlled by simple signals or combinations of signals were subjected to GeneCloud analysis

(https://m2sb.org/?page=AtGeneCloudG).

Table S4. Best marker genes for the simple signals. Pattern matching has been used to uncover

genes mainly regulated by a simple signal as depicted in Fig S6 for the 5 signaling molecules

tested as well as for overall nitrogen content of the media.

Table S4

These genes have been retrieved by Pattern Matching as described in the manuscript.

They represent the best marker genes for the simple signals.

N03- NH4+ IAA CK ABA Nitrogen

At2g15620 At5g07440 At5g54490 At3g50300 At1g53560 At4g37610





At1g80380

At2g23170 At2g47800 At1g20440 At1g22160

At1g49500

At2g33310 At2g22770 At3g50970 At1g27030

At3g25790

At5g47370 At2g01880 At1g77450

At5g62720

At1g59940 At4g19230 At5g67420

At2g25160 At5g57610

At5g13110

At3g48100 At3g48510

At3g29250 At5g06760

At5g50360

At2g37870

At1g02310

At3g61890

At1g08920

At1g20450

At5g27350

At5g11110

At3g51750

At3g17520

At5g04250

At1g02816

At3g14595

At1g77000

At1g24600

https://m2sb.org/?page=AtGeneCloudG

Documents

Supplementary Materials for - Science SignalingSupplementary Materials for Combinatorial interaction network of transcriptomic and phenotypic responses to nitrogen and hormones in