Supplemental Figure 1. Kinetics of seed germination and AtGA20ox3 expression in samples used in Figure 1A. A, Germination kinetics of seeds used in Figure 1A. B, mRNA levels of the AtGA20ox3 gene in samples used in Figure 1A (The AtGA20ox3 gene is under GA negative feed-back regulation; Phillips et al., 1995). Values are relative to ACT8 expression levels. Average and standard errors for two replicates are shown in both panels.

A

B

0

20

40

60

80

100

0 12 24 36 48

Ger

min

atio

n %

Control

GA 50µM

PAC 2µM

Hours after imbibition

0

0,1

0,2

0,3

0,4

0 6 12 24 48

Rel

ativ

e m

RN

A ex

pres

sion

Hours after imbibition (hai)

GA20ox3

NT GA PAC

Supplemental Data. Rombolá-Caldentey et al (2014). Plant Cell 10.1105/tpc114.127647

1

Supplemental Figure 2. Expression of minimal and 4xLIP1 promoter constructs in response to GA. A, Seeds from representative transgenic lines with a minimal (min) or a 4xLIP1 promoter fused to luciferase were imbibed in the absence (NT) or presence of PAC (5 µM) or PAC (50 µM) plus GA (5 µM). Luciferase levels at 48 hai from 20 seeds for each line are shown. B, Pictures of representative luciferase activity observed in 4xLIP:LUC seeds from each treatment.

NT

PAC

PAC+GA

A

B

0

2

4

6

8

10

NT PAC PAC+GA

Luci

fera

se a

ctiv

ity (x

105 )

min

4xLIP1

Supplemental Data. Rombolá-Caldentey et al (2014). Plant Cell 10.1105/tpc114.127647

2

Supplemental Figure 3. GARE occurrences in GA-related Arabidopsis seed transcriptomes and L1-box sequences in the LIP1 and LIP2 promoters. A, The GARE (GA-responsive cis-element) sequence is not enriched in GA-related Arabidopsis seed transcriptomes. Legend as in Figure 3A. B, Alignment of sequences containing the L1-box in the LIP1 and LIP2 promoters.

A

B

0,0

0,2

0,4

0,6

0,8

1,0

1,2

TAIR

GA

up

GA

dow

n

GA

up

DEL

LA d

own

DEL

LA in

dep

GA

dow

n

DEL

LA u

p

DEL

LA in

dep

Aver

age

GA

RE

occu

rren

ces

ga1-3 ± GA WT vs. ga1-3 vs. ga1-3

gai-t6 rga-t2 rgl1-1 rgl2-1

GAup GAdown

-177bp

LIP1

LIP2

CCGCGTGAACATTTAATGCTTCTTCAA

CCGCGTG ACATTTAATGCTTCTTCAA

L1-box core -151bp

-172bp -147bp

Supplemental Data. Rombolá-Caldentey et al (2014). Plant Cell 10.1105/tpc114.127647

3

N-GAI + C-PDF2 N-GAI + C-ATML1

N-ATML1 + C-Ø N-PDF2+ C-Ø

A

B

N-RGA + C-Ø N-GAI + C-Ø

N-Ø + C-ATML1 N-Ø + C-PDF2

N-Ø + C-RGL2

N-PDF2 + C-GAI N-ATML1+ C-GAI

Supplemental Figure 4. BiFC negative assays. A, Absence of interaction between ATML1 or PDF2 and the GAI protein. B, No fluorescence was observed in control experiments coexpressing the ATML1, PDF2 or DELLA proteins with empty vectors (Ø). Overlays of the fluorescence Z-projections and differential interference contrast (DIC) microscopy images are shown.

Supplemental Data. Rombolá-Caldentey et al (2014). Plant Cell 10.1105/tpc114.127647

4

STOP ATG

5´-CAGUUCACAACAUGUAUAAAC-3´

5´-CGGUUCACAACAUGUAUAAAC-3´

3´-GCCGAGUGUUGUACAUAUUUU-5´ amiRNA

STOP ATG

AtML1 cds

PDF2 cds

A

B

C

Supplemental Figure 5. Sequence of amiRTPL-353 and mRNA levels of target genes. A, Sequence of amiRTPL-353 and its corresponding target sequences in the ATML1 and PDF2 genes; B, mRNA levels of ATML1 and PDF2 in amiRTPL-353 lines; C, mRNA levels of HDG2 in amiRTPL-353 lines. Values are relative to ACT8 expression levels and average and standard errors for two replicates are shown in panels B-C.

0

0,02

0,04

0,06

0,08

0,1

0,12

WT 1 2 3 4 5 6

Rel

ativ

e m

RN

A ex

pres

sion

AtML1

0

0,04

0,08

0,12

0,16

0,2

WT 1 2 3 4 5 6

PDF2

0

0,005

0,01

0,015

0,02

0,025

0,03

WT 1 2 3 4 5 6

Rel

ativ

e m

RN

A ex

pres

sion

HDG2

Supplemental Data. Rombolá-Caldentey et al (2014). Plant Cell 10.1105/tpc114.127647

5

0

0,04

0,08

0,12

WT 3 4

Rel

ativ

e m

RN

A ex

pres

sion

LIP1

0

0,01

0,02

0,03

WT 3 4

LIP2

0

0,005

0,01

0,015

0,02

WT 3 4

LTP1

0

0,05

0,1

0,15

0,2

WT 3 4

Rel

ativ

e m

RN

A ex

pres

sion

FDH

0

0,05

0,1

0,15

0,2

WT 3 4

PDF1

0

0,5

1

1,5

2

WT 3 4

MLS

0

0,01

0,02

0,03

WT 3 4

Rel

ativ

e m

RN

A ex

pres

sion

KCS20

0

0,005

0,01

0,015

0,02

WT 3 4

PAS2

0

0,01

0,02

0,03

WT 3 4

Rel

ativ

e m

RN

A ex

pres

sion

ATML1

0

0,01

0,02

0,03

WT 3 4

PDF2

0

0,2

0,4

0,6

WT 3 4

EXP8

0  

0,005  

0,01  

0,015  

WT   3   4  

HDG2

Supplemental Figure 6. GA cannot restore epidermal gene expression in ATML1/PDF2 silenced lines. A, mRNA expression levels of several epidermal and control genes determined in amiRTPL-353 seeds at 24 hai in the presence of GA. Values are relative to ACT8 expression levels and average and standard errors of two replicates are shown.

Supplemental Data. Rombolá-Caldentey et al (2014). Plant Cell 10.1105/tpc114.127647

6

Supplemental Table 1. List of oligonucleotides usedCode Name Locus Sequence (5´- 3´) UseLO273 LIP1-f At5g45670 TTACAAACCCTGCTCGCTACG qPCRLO274 LIP1-r At5g45670 TGGTGCTTGACCTGGAAGACA qPCRLO277 Malatesynth-f At5g03860 AACCGCTAAGCTTTTTGTCCG qPCRLO278 Malatesynth-r At5g03860 GGCCAAAATCGACAAGACATC qPCRLO1106 AtGA20ox3-f At5g07200 ATTACTACCCGCAGTGCAAGCA qPCRLO1107 AtGA20ox3-r At5g07200 ACCGCCAACTTGGTCTTGATG qPCRLO1258 ATML1-f At4g21750 AGCATGCTGAAACTAGCGGAGA qPCRLO1259 ATML1-r At4g21750 TCGGATCCTGTGGTCGACAAT qPCRLO1340 PDF2-f At4g04890 AAGCCGTTAGGATCGTCTTTCG qPCRLO1341 PDF2-r At4g04890 CCTACAAAGCCTGTCTGGTTCC qPCRLO1342 attB1-PDF2 At4g04890 AAAAAAGCAGGCTTCATGTACCATCCAAACATGTTTG pGADT7:PDF2LO1343 attB2-PDF2 At4g04890 CAAGAAAGCTGGGTCCTACGCTCCTCCTCCAACATC pGADT7:PDF2LO1496 LIP1probe-f At5g45670 CTGCTTCTGATGCTCATCC LIP1 probeLO1497 LIP1probe-r At5g45670 GAATCAAATTTATAAAACTG LIP1 probeLO1500 prom ΔLIP-rv At5g45670 GGATCCGTAAAAGTAAATGTGATG Promotermut:GUS (pBT10)LO1501 prom ΔLIP-fw At5g45670 GGATCCCTTTATAAAATAACAG Promotermut:GUS (pBT10)LO1502 promLIP-fw XbaI At5g45670 TCTAGAAACATCATTATTCACCGTTG Promoter:GUS (pBT10)LO1503 promLIP-rv-NcoI At5g45670 CCATGGTCATCAAACTCATTCTCGCCAT Promoter:GUS (pBT10)LO1576 S-AtMAN7 (RT2) - f At5g66460 TCAGGGACAAGTTGGGAAAC qPCRLO1577 AS-AtMAN7 (RT2)- r At5g66460 AGCTGAGATCGTTCCATTCG qPCRLO1772 ACT8-f At1g49240 GGTCGTACAACCGGTATTGT qPCRLO1773 ACT8-r At1g49240 GAAGAGCATACCCCTCGTA qPCRLO1776 PDF1-f At2g42840 TGGAGAAACCACCCTACATTGA qPCRLO1777 PDF1-r At2g42840 CAAACCCTGGAATGCTACTGG qPCRLO1778 FDH-f At2g26250 TAGCATTTTCAACCCGACACC qPCRLO1779 FDH-r At2g26250 AACATCCCATCCCTCCAAGGT qPCRLO1780 LTP1-f At2g38540 TGAAGTTGGCATGCTTGCTCT qPCRLO1781 LTP1-r At2g38540 CGCTGCCAAGTTGCTGTTAAC qPCRLO1782 ACR4-f At3g59420 ATAAACGCACTCTTGGACCCG qPCR

LO1783 ACR4-r At3g59420 TTCCCTCTCATTCTCACGCAT qPCRLO1784 LIP2-f At4g18970 TAAACCGCTACACCGAGCAAC qPCRLO1785 LIP2-r At4g18970 CAGCTCATTTGGACTGCATCC qPCRLO1797 HDG2-F At1G05230 CATGCCTTTGGTGCTAAACGC qPCRLO1798 HDG2-R At1g05230 ACGCCAACTTCTCCAGAGGAA qPCRLO1803 EXP8-F At2g40610 GCTCAAAAACACAGTCGTGGC qPCRLO1804 EXP8-R At2g40610 CGTTACCTGGAAGGAAAGGCT qPCRLO1809 PAS2-F At5g10480 CTGCCACAGATAGGTTCAAGGC qPCRLO1810 PAS2-R At5g10480 CAGCTTATGACCAGCGACGTAA qPCRLO1811 KCS20-F At5g43760 GCACTCGTGAAACATTCATGGA qPCRLO1812 KCS20-R At5g43760 ACCTAGACCGGATCTTTCGAGG qPCRLO1827 Fw-IS-ML1 At4g21750 CCGGATTCGCTATTTTGCCG ATML1 probeLO1828 Rv-IS-ML1 At4g21750 ACATCGATTAGGCTCCGTCG ATML1 probeLO1829 Fw-IS-PDF2 At4g04890 CGTTGTTGCCGTCTGGTTTT PDF2 probeLO1830 Rv-IS-PDF2 At4g04890 CGCTCCTCCTCCAACATCAC PDF2 probe