6

4

0

2

8C

oty

led

on

are

a (

mm

2)

A

Le

r

sp

t-12

sp

t-1

sp

t-2

sp

t-3

sp

t-11

Co

l

B

Exp

ressio

n r

ela

tive

to C

SY

3

WT

35

S:

SP

T

0

10

20

30

Hyp

oco

tyl le

ngth

(m

m)

8

4

0

Co

l

sp

t-11

pif4

-2

pif4

-10

1

pif3

-3

pif7

-1

C

Co

tyle

don

are

a (

mm

2)

0 0

Col spt-11 spt-12 Col spt-11 spt-12

15ºC 25ºC

0

2

4

8

10

6

D

Supplemental Figure 1. Characterisation of spt alleles, pif alleles and 35S:SPT seedlings.(A) Cotyledon area of 7-day old Col (WT), spt-11, spt-12, Ler (WT), spt-1, spt-2, spt-3 seedlings grown under red light at 20ºC. (B) SPT mRNA levels (relative to CSY3 expression) is shown for 35S:SPT transgenic and Col WT plants used in Figure 1B and 4A, as measured by quantitative real-time PCR. Values ± SE were obtained from three biological replicate experiments. (C) Hypocotyl length of 12-day old Col, spt-11, pif4-2, pi4-101, pif3-3 and pif7-1 seedlings grown under red light at 20ºC.(D) Cotyledon area of 12-day old (left) or 7 day-old (right) seedlings grown under red light at either 15ºC or 25ºC. Seedlings grown at 15°C experienced an extended growth period to bring seedlings to a similar developmental age. In all graphs, bars indicate SE.

Supplemental Data. Josse et al. (2011). Plant Cell 10.1105/tpc.110.082594

WT spt-11 spt-12

0

1,000

2,000

3,000

4,000

5,000

6,000

Ce

ll a

rea

(µ

m2)

*

**

WT spt-11 spt-12

0

500

1,000

1,500

2,000

2,500

3,000

Ce

ll pe

rim

ete

r (µ

m)

***

Supplemental Figure 2. spt mutants exposed to GA have larger cotyledon pavement cells than the wild type. Box plots for cell area and cell perimeter measured from three independent SEM images per genotype (as pictured in Figure 1F) each containing at least 90 complete cells. The median, lower quartile, upper quartile, as well as the minimum and maximum for each population are represented. The Kolmogorov-Smirnov test shows that GA-treated spt-11 and spt-12 mutant contain cells with larger areas and longer perimeters than GA-treated WT seedlings, with a confidence level of 99% (**) and 95% (*) respectively. (p values for cell area: 0.002 (spt-11), 0.048 (spt-12); p values for cell perimeter: 0.003 (spt-11), 0.026 (spt-12).

Supplemental Figure 3. The Ler and Col accessions respond to GA differently.

(A) Cotyledon area of 8-day old WT seedlings grown under red light at 20ºC. Seedlings were either untreated (left) or PAC (0.2µM) treated (right). Seedlings either received no GA3

(white columns) or 1µM GA3 (black columns).In all graphs, bars indicate SE.(B) Cotyledon area of 8-day old Ler, spt-2 and spt-3 seedlings grown under red light in untreated controls and in the presence of increasing concentrations of GA3.

6

5

4

3

2

1

0Ler Col Ler Col

PAC treateduntreated

A no GA

+ GA

0 1 5 50

GA concentration (µM)

6

4

0

2

8B

spt-2

spt-3

Ler WT

Supplemental Data. Josse et al. (2011). Plant Cell 10.1105/tpc.110.082594

Supplemental Figure 4. Cotyledon-located GFP-RGA accumulates in the dark. A representative 4-day old dark-grown seedling (expressing pRGA:GFP-RGA) is presented, showing accumulation of GFP-RGA in the cotyledon, and absence of GFP-RGA in the hypocotyl.

Supplemental Data. Josse et al. (2011). Plant Cell 10.1105/tpc.110.082594

Supplemental Figure 5. RGA accumulation in the cotyledon is controlled by phytochrome B.GFP-RGA detection by confocal microscopy in the cotyledons of 12-day old seedlings expressing pRGA:GFP-RGA in a phyB-9 (A) or a WT (B, C) genetic background. Seedlings were grown at 20ºC under red light [2µmol.m-2.s-1; R:FR>2] (A and B), or under red light supplemented by far-red light [2µmol.m-2.s-1; R:FR<0.1] (C). Corresponding bright-field images are provided to allow a visual comparison of the cell sizes in the different conditions (D,E,F).

WTRED

(R:FR>2)

WTRED + FAR-RED

(R:FR<0.1)phyB

RED

A B C

D E F

-2h

+ 3

0m

in

+ 1

h

+ 2

h

+ 4

h

+ 8

h

SPT-HA

loading

Supplemental Figure 6. SPT protein levels are stable in red light.

SPT-HA levels detected by immunoblot in 5-day old seedlings grown under 12h red / 12h dark photoperiods at 15ºC. Sample times are as indicated. Coomassie staining of a twin gel is used as a loading control.

Supplemental Data. Josse et al. (2011). Plant Cell 10.1105/tpc.110.082594

[bait] BD-GAL4x

[prey] AD-GAL4

Ø = empty vector

Øx

SPT

SPTx

SPT

SPTxØ

ØxØ

1

+

+

+

+

SPTx

RGA

Øx

RGA

SPTx

GAI

Øx

GAI

132

18

164

1 132

18

164

-

-

-

-

+

+

+

+

-

-

-

-

Supplemental Figure 8. SPT interacts with RGA and GAI in a Yeast-Two-Hybrid assay.Interaction of SPT with RGA and GAI shown by yeast-2-hybrid experiment. Dilution spots of yeast strains expressing ORF-BD-GAL4 and ORF-AD-GAL4 (as indicated) grown on SD media (+) or SD media without His and Ade (-). BD: binding domain, AD: activation domain.

SPT, RGA and GAI full cDNAs were cloned using the Gateway® technology into pDONOR 207. The pENTRY vector obtained were subsequently used to introduced SPT into pDEST32 (GAL4 activation domain fusion) and GAI and RGA inserts into pDEST22 (GAL4 DNA-binding fusion) (InvitrogenProQuestTM Two-Hybrid System). Relevant plasmids (including control empty pDEST 22 and pDEST 32) were co-transformed in the yeast strain AH109 and transformants were selected on SD media depleted of Leucine and Tryptophan. Protein interaction was tested by the ability of the colonies to grow on SD depleted of Tryptophan, Leucine, Adenine and Histidine.

BASIC HELIX I LOOP HELIX II

* * ** * **

DNA-binding DNA backbonedimerisation

*

**

SPT KRCRAAEVHNLSEKRRRSRINEKMKALQSLIPNSNKTDKASMLDEAIEYLKQLQLQVQ

PIF3 KRSRSAEVHNLSERRRRDRINEKMRALQELIPNCNKVDKASMLDEAIEYLKSLQLQVQ

PIF4 RRSRAAEVHNLSERRRRDRINERMKALQELIPHCSKTDKASILDEAIDYLKSLQLQLQ

Supplemental Figure 7. SPT, PIF3 and PIF4 share a highly conserved bHLH domainProtein sequence alignment of the bHLH domain from PIF3, PIF4 and SPT. Important domains and residues are noted.

Supplemental Data. Josse et al. (2011). Plant Cell 10.1105/tpc.110.082594

GA [50µM]GFP-RGA

UGPase

0 1h� h �h 1½h 8h3h 4h 2h

GFP-RGA

UGPase

GFP-RGA

UGPase

↓↓↓↓

GA [10µM]

GA [1µM]

GA

Supplemental Figure 9. Dose-dependent GA-mediated GFP-RGA degradation GFP-RGA accumulation in 6-day old red-light grown seedlings detected by western blot. Seedlings harvested at the times shown (in hours), post GA3 application (at the concentration indicated to the left of each blot). UGPase detection was used as a loading control.

Supplemental Data. Josse et al. (2011). Plant Cell 10.1105/tpc.110.082594

12.660475Suppl. dataset 10Zentella et al.,

2007

RGA-regulated genesShoots of 8-d-old seedlings, grown under WL (100µmol.m-2s-1) at 22ºC on MS +2% suc

13.0104802Suppl. dataset 9Hou et al., 2008RGA-regulated genesInflorescence apices containing floral buds younger than stage 10

11.673632Suppl. dataset 8Cao et al., 2006DELLA-regulated genesUnopened buds from 28-d-old WT, 22d-old ga1-3 della quad mutant and 38-d-old ga1-3 mutant

13.985610Suppl. dataset 7Cao et al., 2006DELLA-regulated genesSeeds imbibed on filter paper soaked in sterile water for 4 days, Wc, 4ºC

18.399542Suppl. dataset 6Achard et al.,

2008

DELLA-regulated genes12-d-old seedlings, grown on MS (solid MS for 9 days, liquid MS for 3 days) ± NaCl 200mM

6.2152424576 Suppl. dataset 1----Arabidopsis ATH1 Genome Array

% SPT-regulated

genes

Genesscored as

SPT-regulated

Total number of

genes

SPT-regulated Gene List

ReferenceSpecification Tissue / experimental conditions

20.93191524Suppl. dataset 11Suppl. dataset 1SPT-regulated genes

4-d-old seedlings grown on Gilroy medium (no suc) under RL (30µmol.m-2s-1), 20ºC ± GA 50µM

10.8265224576----Suppl. dataset 1Arabidopsis ATH1 Genome Array

% DELLA-regulated

genes

Genesscored as DELLA-

regulated

Total number of

genes

List of DELLA-regulated genes

SPT-regulated Gene List

Specification Tissue / experimental conditions

A

B

Supplemental Figure 10. SPT and DELLAs regulate a common gene subset.(A) Table presenting the number of SPT regulated genes previously identified as DELLA-regulated in published micro-array data. Our analysis using the Arabidopsis ATH1 genome array identified 1524 SPT controlled genes, indicating that ~6.2% of the genome was subject to SPT regulation. The proportion of SPT-regulated genes is significantly enriched in genes identified previously as DELLA targets (between 11.6% and 18.3%, χ2 p<0.0001 for the 5 datasets presented). (B) Table presenting SPT-controlled genes from our study that were also identified as DELLA-regulated in published micro-array data. The total DELLA targets number (2652 genes) discounts duplicated genes amongst the 5 published reference datasets (shown in A); this indicates that 10.8% of the Arabidopsis ATH1 genome array is DELLA-regulated. There is a significant enrichment for DELLA-regulated genes (20.9%, χ2 p<0.0001). amongst the SPT-controlled genes identified in our study.

Supplemental Data. Josse et al. (2011). Plant Cell 10.1105/tpc.110.082594

Supplemental Figure 11. SPT and DELLAs mostly regulate genes in a similar direction.Pie charts representing the proportion of DELLA-regulated genes (identified in earlier studies) within the SPT-regulated subset (shown in red). For 29% of these genes, SPT and DELLAs had opposing regulatory control, while for 64% of these genes, SPT and DELLAs imposed the same directional control. For a small proportion (7%) of these genes, shown in white, we could not detect a consistent regulatory pattern across the published data. Gene lists are presented in supplemental datasets 6-11. SPT regulated genes were determined as gene presenting at least a 1.5 fold change in expression between WT and spt-12. In our study, mean values were derived from three biological replicate experiments. DELLAs regulated genes were extracted form the micro-arrays analyses published in: Achard et al., 2008; Cao et al., 2006; Hou et al., 2008 and Zentella et al., 2007.

Genes regulated by both SPT and DELLAs

Genes regulated in a similar direction by SPT and DELLAs

Genes regulated in opposite directions by SPT and DELLAs

Genes with no consistent regulatory patterns across published data-sets

Genes regulated by SPT only

Genes presenting at least a 1.5 fold change in expression

between WT and spt-12(1524 genes)

319 genes(21%)

203 genes(64%)

92 genes(29%)

Supplemental Data. Josse et al. (2011). Plant Cell 10.1105/tpc.110.082594

CTATTGAACACTAGACACCACTTGCTTCTTACTCCTTAGCAACGACAT3G63010GID1b

AAACAACCTTCTTTCTCTAACCCACCTCTTGCCTAATAACAATCATTAT3G05120GID1a

CCTCTCATTACTGCCTCAACGTCCTGCGATATTTAGATCATTGTAT1G67100LBD40

GCTTGTTGATTCTTTCAGGTTGATATTGGTTGCATCGTTTCTGAT2G45900

TTTATCAAGGGAAAGAACAGAAGTTCAGGAAGAGGGAGAAGAAAGAT5G50915BHLH137

AACACGTCCTGCTTCAATATCTTAAAGTTGCTTCCATGATCCCAT3G11280MYB

GAAAGCTGATGGTCTTCTGGATGCTTGGAGAAATGGATAGACAT2G04240XERICO

TAGCAAGAGGTGGATTAAGTAGATATGTCACTCAACAGACCGAAT1G50240SCL3

ATCGTATGGGTAACTAGTCCCGTCATTCCAACGCAAACATAACAT4G36930SPT-HA

GAAACAAGAACACATAGAGATCCCGTCATTCCAACGCAAACATAACAT4G36930native SPT

TGAACAATCGATGGACCTGACAGTGTCTGGATCGGAGGATAT5G09810ACT7

TGGTCTTTCCGGTGAGAGAGTCTTCACACTCCACTTGGTCTTGCGTAt4G05320UBQ10

primer 2primer 1Gene IDGene name

Supplemental Table 1: List of the primers used in the qPCR experiments

Supplemental Data. Josse et al. (2011). Plant Cell 10.1105/tpc.110.082594