Supplemental Figure 1. Relative accumulation of WRI mRNAs in siliques of transgenic lines. Analyses of the relative WRI mRNA levels were performed by qRT-PCR on siliques aged 16 days after anthesis. The results obtained were standardized to the constitutive EF1αA4 gene expression level (EF). For each construct considered, two independent overexpressing lines were analyzed. For each genotype, three independent cDNA preparations were obtained from independent individuals; one or two technical replications were realized with each cDNA preparation analyzed. Values are the means and SE of between three and six measurements.

Rel

ativ

e m

RN

A le

vel (

% E

F)

ProAT2S2:WRI1 in wri1-4

ProAT2S2:WRI2 in wri1-4

ProAT2S2:WRI3 in wri1-4

ProAT2S2:WRI4 in wri1-4

WRI1 WRI2 WRI3 WRI4

1

Supplemental Data. To et al. Plant Cell. (2012). 10.1105/tpc.112.106120

Supplemental Figure 2. Expression of genes involved in fatty acid modification in maturing seeds of wri1 wri3 wri4 mutants. The expression patterns of genes encoding prominent enzymes involved in fatty acid modification in the endoplasmic reticulum were investigated by qRT-PCR and presented as percentage of the constitutive EF1α4 (EF) gene expression. Values are the means and SE of three replicates carried out on three independent cDNA preparations obtained from batches of seeds dissected from 4 to 5 siliques. The three silique sets were harvested on distinct individuals. WT, wild type (Col-0).

Rel

ativ

e m

RN

A le

vel (

% E

F)

Rel

ativ

e m

RN

A le

vel (

% E

F)

1.4 1.4 1.4 3.1 3.1 3.2 4.1 4.3 4.3

WT

wri1 wri3 wri4

1.4 1.4 1.4 3.1 3.1 3.2 4.1 4.3 4.3

WT

wri1 wri3 wri4

KCS18/FAE1

FAD3

FAD2

ROD1

2

Supplemental Data. To et al. Plant Cell. (2012). 10.1105/tpc.112.106120

Supplemental Figure 3. Complementary results for the characterization of the flower phenotype of wri mutants. (A) Percentages of unopened flowers and aborted siliques on inflorescence stems of wild-type and mutant lines. Values are the means and SE of observations carried out on 5 different plants. On average, 100 to 150 flowers were observed for each plant. (B) Permeability to toluidine blue of the epidermis of petals and sepals of wild-type and mutant lines. Representative flowers are presented.

A

%

Unopened flowers

Aborted siliques

B

1-4 WT 3-1

4-3

1-4 1-4 1-4 3-1 3-1 3-1

4-3 4-3 4-3

1-4 WT 3-2

4-3

1-4 1-4 1-4 3-2 3-2 3-2

4-3 4-3 4-3

Unopened flowers

Aborted siliques

wri mutants wri mutants

3

Supplemental Data. To et al. Plant Cell. (2012). 10.1105/tpc.112.106120

Supplemental Figure 4. Cutin analyses of inflorescence stems and rosette leaves of the wri1-4 wri3-1 wri4-1 mutant. Amounts of cutin monomers are expressed on a dry weight basis of delipidated material. Each constituent is designated by carbon chain-length and labeled by chemical class along the x-axis. Values are the means and SE of three replicates carried out on different plants. .

µg.m

g-1 D

W

Col-0 wild type wri1-4 wri3-1 wri4-1

Dicarboxylic acids 2-Hydroxy acids ω-Hydroxy acids Fatty acids

Inflorescence stems

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

C16:0 C18:0 C20:0 C24:0 C26:0 C16:0 DCA

C18:0 DCA

C18:1 DCA

C18:2 DCA

C18:1 ωOH

C18:2 ωOH

20H C16:0

20H C22:0

20H C23:0

20H C24:0

20H C24:1

20H C25:0

20H C26:0

µg.m

g-1 D

W

Col-0 wild type wri1-4 wri3-1 wri4-1

Dicarboxylic acids 2-Hydroxy acids ω-Hydroxy acids Fatty acids

Rosette leaves

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

C16:0 C18:0 C20:0 C24:0 C26:0 C16:0 DCA

C18:0 DCA

C18:1 DCA

C18:2 DCA

C18:1 ωOH

C18:2 ωOH

20H C16:0

20H C22:0

20H C23:0

20H C24:0

20H C24:1

20H C25:0

20H C26:0

0.40

4

Supplemental Data. To et al. Plant Cell. (2012). 10.1105/tpc.112.106120

Supplemental Figure 5. Cuticular wax composition of wild-type and wri1 wri3 wri4 flowers. Amounts of major components are expressed as ng.mg-1 of fresh weight. Each wax constituent is designated by carbon chain-length and is labeled by chemical class along the x-axis. Values are the means and SE of three replicates carried out on different plants. 2°-Alc, secondary alcohols; Ket, ketones. .

ng.m

g-1 D

W

Ket. Aldehydes Esters Alkanes

0

100

200

300

400

500

Col-0 wild type wri1-4 wri3-1 wri4-1 wri1-4 wri3-1 wri4-3 wri1-4 wri3-2 wri4-3

2°-Alc. 1°-Alcools

25 27 29 31 33 i29 29 29 24 26 28 30 24 26 28 30 38 40 42 44

5

Supplemental Data. To et al. Plant Cell. (2012). 10.1105/tpc.112.106120

Supplemental Figure 6. WRI relative mRNA levels of wri mutant floral organs. Analyses of the relative WRI mRNA levels were performed on petals and sepals of opening flowers by qRT-PCR. The results obtained were standardized to the constitutive EF1αA4 (EF) gene expression level. Three independent cDNA preparations were analyzed; one or two technical replications were carried out with each cDNA population prepared. Values are the means and SE of between three and six measurements.

WRI1 Rel

ativ

e m

RN

A le

vel (

% E

F)

WRI3 WRI4

wild type (Col-0)

wri1-4

wri3-1

wri4-1

6

Supplemental Data. To et al. Plant Cell. (2012). 10.1105/tpc.112.106120

Supplemental Data. To et al. Plant Cell (2012). 10.1105/tpc.112.106120 WRI3 PQRSSVHRGV TRHRWTGRYE AHLWDKNSWN ETQTKKGRQV YLGAYDEEDA WRI4 LQRSSPYRGV TRHRWTGRYE AHLWDKNSWN DTQTKKGRQV YLGAYDEEEA WRI1 TRRSSIYRGV TRHRWTGRFE AHLWDKSSWN SIQNKKGKQV YLGAYDSEEA WRI2 GKRSSIYRGV TRHRWTGRYE AHLWDKSTWN QNQNKKGKQV YLGAYDDEEA AP2#1 WRI3 AARAYDLAAL KYWGRDTILN FPLCNYEEDI KEMESQSKEE YIGSLRRKSS WRI4 AARAYDLAAL KYWGRDTLLN FPLPSYDEDV KEMEGQSKEE YIGSLRRKSS WRI1 AAHTYDLAAL KYWGPDTILN FPAETYTKEL EEMQRVTKEE YLASLRRQSS WRI2 AARAYDLAAL KYWGPGTLIN FPVTDYTRDL EEMQNLSREE YLASLRRKSS WRI3 GFSRGVSKYR GVAKHHHNGR WEARIGRVFG NKYLYLGTYA TQEEAAIAYD WRI4 GFSRGVSKYR GVARHHHNGR WEARIGRVF- ---------A TQEEAAIAYD WRI1 GFSRGVSKYR GVARHHHNGR WEARIGRVFG NKYLYLGTYN TQEEAAAAYD WRI2 GFSRGIAKYR GLQS-----R WDASASRMPG PEYFSNIHYG AGDDRGTEGD AP2#2 WRI3 IAAIEYRGLN AVTNFDISRY L WRI4 IAAIEYRGLN AVTNFDVSRY L WRI1 MAAIEYRGAN AVTNFDISNY - WRI2 FLG----SFC LERKIDLTGY I    Supplemental Figure 7. Alignment of amino acid sequences corresponding to the DNA-binding domains of WRI1, WRI2, WRI3, and WRI4. The alignment was generated by the MUSCLE program at phylogeny.fr. The two AP2 motifs are underlined and termed AP2#1 and AP2#2. Amino acid residues are colored as follows: red: high consensus; blue: low consensus; black: non-conserved residue.

7

Supplemental Figure 8. Characterization of WRI2. (A) Analyses of WRI2 relative mRNA accumulation levels were performed by qRT-PCR with different plant organs and developing siliques. The results obtained were standardized to the constitutive EF1αA4 (EF) gene expression level. Values are the means and SE of three to four replicates carried out on cDNA dilutions obtained from three independent mRNA extractions. DAA, days after anthesis. (B) Molecular characterization of the wri2 mutations. Structures of the WRI2 gene showing the position of T-DNA insertions in wri2-1 and wri2-2 are presented. For each T-DNA insertion considered, confirmed flanking sequence tag(s) are anchored in the gene structure and represented by vertical bar(s). Closed boxes represent exons while open boxes stand for untranslated regions (UTRs). (C) Accumulation of WRI2 mRNA was quantified by qRT-PCR with siliques (aged 12 days after anthesis) of WRI2 overexpressing lines and wri mutants. Results are presented as percentage of the constitutive EF1α4 (EF) gene expression. Values are the means and SE of three replicates carried out on three independent cDNA preparations obtained from batches of 4 to 5 siliques. The three silique sets were harvested on distinct individuals. ND, not detected. (D) The expression levels of two fatty acid biosynthetic genes, BCCP2 and PKp2 were investigated in developing siliques aged 12 days after anthesis in WRI2 over expressing lines and in wri mutants by qRT-PCR. Results are presented as percentage of the constitutive EF1α4 (EF) gene expression. Values are the means and SE of three replicates carried out on cDNA dilutions obtained from three independent mRNA extractions. (E) The fatty acid concentrations of dry seeds obtained from WRI2 overexpressing lines and wri mutants were determined by GC analysis. Values are the means and SE of five replicates carried out on batches of 20 seeds from five distinct individuals. T9R4, T12R3, ProS5Sdual:WRI2 independent lines; WT, wild type (Col-0).

B

C

E

T12R

3

WT

T9R

4

wri2

-1

wri2

-2

wri1

-4

wri1

-4 w

ri2-2

See

d fa

tty a

cid

cont

ent

(µg.

mg-

1 D

W)

wri2

-1

wri2

-2

WRI2 – At2g41710

ATG

Rel

ativ

e W

RI2

m

RN

A le

vel (

% E

F)

ND ND ND

Rel

ativ

e m

RN

A le

vel

(% E

F)

D

T12R

3

WT

T9R

4

wri2

-1

wri2

-2

wri1

-4

wri1

-4 w

ri2-2

T12R

3

WT

T9R

4

wri2

-1

wri2

-2

wri1

-4

wri1

-4 w

ri2-2

BCCP2 PKp2

A

Rel

ativ

e W

RI2

m

RN

A le

vel (

% E

F)

Siliques (DAA)

8

Supplemental Data. To et al. Plant Cell. (2012). 10.1105/tpc.112.106120

Supplemental Figure 9. Version of the phylogram among AP2 transcription factors of the APETALA, AINTEGUMENTA, and WRINKLED clades presenting statistical support for nodes. Phylogram, with branch lengths in arbitrary units, using the alignment generated by the MAFFT program. Sequences of the double AP2 (DNA-binding) domains of the transcription factors (with gaps) were used for the distance analyses. Percentage values (in red) on each branch represent the corresponding bootstrap probability.

WR

I1-like

At3g20840

At1g51190

0.01

At5g65510

At5g10510

At3g54990

At2g39250

At5g60120

At2g28550

At4g36920

At5g67180

WRI1/At3g54320

WRI3/At1g16060

WRI4/At1g79700

At5g57390

At5g17430

At4g37750

At1g72570

WRI2/At2g41710

100

31

98

100

100

62

54

99

45

19 55

73 82

91

96

62

AIN

TEG

UM

EN

TA-like

AP

ETA

LA-like

9

Supplemental Data. To et al. Plant Cell. (2012). 10.1105/tpc.112.106120

Supplemental Data. To et al. Plant Cell. (2012). 10.1105/tpc.112.106120

Supplemental Table 1. Primers used for quantitative RT-PCR

Gene

AGI

Forward primer (5’3’)

Reverse primer (5’3’)

BCCP2 At5g15530 GACCCGGTGAACCCCCT GTCAACGCTGACTGGTTTTCCAT

CYP77A6 At3g10570 TCGGGTAAGGAGGAAGCGGAC CGCTCCACTCAAACTCTTGAACCAT

CYP86A2 At4g00360 GAGAAACGACGTCGTTGTTCTTAACG AACCCGAGAAGCTGCATTACACGG

CYP86A8/LCR At2g45970 TGAAATCGACTTGGGGAGAG GCCAGATCCTTACCCAGACA

DAGAT1 At2g19450 Quantitect Primer Assay (Qiagen) EF1alphaA4 At5g60390 CTGGAGGTTTTGAGGCTGGTAT CCAAGGGTGAAAGCAAGAAGA

ENR At2g05990 TGGGACTTGGGTTCCTGCAC CGCTTATTCGTTTTCACATCTTCAGGC

FAD2 At3g12120 CCGTCCAGTTTGTCCTCGGG CGGTAAAGACCAAAACAGACG

FAD3 At2g29980 CATCGCTGCCGTGTATGTTG GGAACCCATGACTCGTCGTT

FAE1/KCS18 At4g34520 CCAAGTACAAGCTAGTTCACACG ACTCCGATTTTGCCGCTCT

FATA At3g25110 ACAACAACTACTATGAGGAAGTTGCATCT TCCAATCACGCCTTGTCCCA

GPAT4 At1g01610 TCCTGAAGGCACAACGTGTA ACATCCCTTGCTTGCAGTTC

GPAT6 At2g38110 TTGGTGGCCTAGCGTCTGAT GCGTTGGGCGTTGGACT

GPAT8 At4g00400 AATGACTGTCAACGGTGGTGGCAAG GCGAGTAAGTTCGGTGCATTCGAAG

GPAT9 At5g60620 GCCTCATGGACCGGAGTTGT CCAACCCAACCAGGATGCT

GPDH At3g07690 TGTGGAAGCAGAGTTTGAGCCT ACCCGCAAATCCGTGCAT

GPDHc1 At2g41540 GGGAGGTCTCAAGAATGTCTACGC AGCAAAGGCCCTGCAAGTT

KAS1 At5g46290 GGGTTCTGCTTTGTTGGCGA GCCTCAGTCCCACCAGCAAT

KASIII At1g62640 TCTGTGGCTACAAGGCTGCAT TGCTGATCCCCACGTTAAACCG

LPAAT4 At1g75020 ACGAGGCAGAATCTTCTGCTTGG CTGTCAAAGATATTACCCCTGCAAATGT

MAT At2g30200 CATGGTTAGTATCATAGGGTTGGACTCAGA TGGCTTCAACAACTTCGATTCCTTTAAGA

PDAT1 At3g44830 CGTAGTCAACATTGGACCCGCC TCCCAGCTATGTGACATCCGC

PDH E1a At1g01090 ATGTGTGCTCAAATGTATTACCGAGGC ACCTTTGCTGAGGGCATGG

PKp2 At5g52920 AGTCACTATCGTCCTTCCG CTGTACGATTGCTATTTCCTC

ROD1 At3g15820 GGTGTCGGAGGAGGAGGAT CGTGTACTCCACGCCCATGA

WRI1 At3g54320 Quantitect Primer Assay (Qiagen)

WRI2 At2g41710 Quantitect Primer Assay (Qiagen)

WRI3 At1g16060 Quantitect Primer Assay (Qiagen)

WRI4 At1g79700 ATAATGGGAGATGGGAAG CGGCGATGTAGATGG

10