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. Relative mRNA level (% EF) 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
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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
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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. .
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. .
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
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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
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RI2
m
RN
A le
vel (
% E
F)
ND ND ND
Rel
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RN
A le
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(% 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
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RI2
m
RN
A le
vel (
% E
F)
Siliques (DAA)
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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