Supplemental Data. Hachez et al. Plant Cell (2014) /tpc Suppl. Figure 1A

Transcription

1 Suppl. Figure 1A

2 Suppl. Figure 1B Supplemental Figure 1: Results of the commercial screening of interactants using split ubiquitin technique. (A) Isolated preys (192) using the bait construct pbt3-n- as described in Figure 1, and their corresponding β-galactosidase activity with time. Circled in red are the selected clones (35) showing the strongest β-galactosidase activity. (B) Isolated preys (43) using the bait construct pbt3-suc- as described in Figure 1, and their corresponding β- galactosidase activity with time. Circled in red are the selected clones (10) showing the strongest β-galactosidase activity.

3 Transcriptional regulation during seed development: PIP2;7 Supplemental Figure 2: Comparative transcriptional regulation of Arabidopsis and PIP2;7 Developmental expression pattern of At4g35100 (encoding PIP2;7, upper histogram) and At2g47770 (encoding, bottom histogram), from transcriptome data as compiled and analyzed by Winter et al. (2007) accessible at b.cgi. As detailed in the right panel, the expression of PIP2,7 and at the transcript level appears to be mutually exclusive during seed development. Suppl. Figure 2

4 A PIP2;7 WT Supplemental Figure 3: Time course expression of PIP2;7 and during seed imbibition and germination (A) Total proteins were extracted from dry wild-type seeds (lane 1) or from germinating seeds incubated for 1 day (lane 2), 2 days (lane 3), 3 days (lane 4) or 4 days (lane 5), and subjected to immunoblotting. The blot were probed for PIP2;7,, and actin as loading control. (B) The same extracts as in (A) but from a TDNA insertional knockout line for 40 Actin B PIP2;7 KO kda Actin

5 A kda B IN YFP- FT W E kda 21 IN 21 E IN PMA E 40 YFP Supplemental Figure 4: Technical controls for Figure 2 (A)Proteins extract from Arabidopsis wild-type dry seeds or transgenic lines expressing YFP-PIP2;7 or ST-GFP was affinity purified using GFP-Trap. The input (IN), and eluted fractions (E) were analyzed by immunoblotting and probed for or the plasma membrane H+-ATPase (PMA). Pulldown of was mainly evident with seeds expressing YFP-PIP2;7 (B) Proteins extract was prepared from transgenic Arabidopsis seedlings overexpressing YFP- and the fusion protein affinity-purified using GFP-Trap. The input (lane IN), the flow-through (lane FT), the last wash (lane W, wash #3), and eluted fractions (lane E) were analyzed by immunoblotting for or GFP/YFP.

6 A YFP-PIP2;7 in OE8 (T2) B YFP-PIP2;7 in OE8 (T2) Supplemental Figure 5: Expression pattern of YFP-PIP2;7 in segregating population coexpressing. C YFP-PIP2;7 in OE9 X (T2) OE9 (F2) D YFP-PIP2;7 in OE9 (T2) Homozygote independent transgenic lines (OE8 and OE9) overexpressing At- were retransformed to express YFP-PIP2;7. T2 seedlings from the same batch were scored for hygromycin resistance and YFP fluorescence pattern. Confocal images of cotyledons from T2 individuals showed patchy YFP fluorescence (55/99 seedlings) (green channel, panels A and C, magenta channel, chlorophylls) or no YFP expression (25/99 seedlings) for seedlings resistant to hygromycin (selection marker in OE8 and OE9) (80/99 seedlings); distribution of YFP fluorescence (grey channel, panels B and D) for seedlings sensitive to hygromycin (19/99 seedlings) Bars 20 µm.

7 PIP2;7::YFP-PIP2;7 Supplemental Data. Hachez et al. Plant Cell (2014) /tpc UBI10::RFP- PIP2;7::YFP-PIP2;7 + UBI10::RFP- YFP Supplemental Figure 6: UBI10-driven expression of RFP- shows a patchy distribution of RFP fluorescence. Wild-type Arabidopsis or a transgenic line expressing YFP-PIP2;7 (under the control of its endogenous promoter) were transformed to express RFP tagged under the control of the Arabidopsis constitutive promoter of ubiquitin 10. Confocal imaging showed that in the WT background, RFP- fluorescence was patchy and induced a patchy distribution of YFP-PIP2;7 when coexpressed. Bar = 20 µm, BF = bright field. RFP BF Merge

8 Relative water lost Relative water lost Supplemental Data. Hachez et al. Plant Cell (2014) /tpc A B 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0, Time (min) WT KO OE8 Supplemental Figure 7: Relative water lost from various mutants (A) Water lost with time from detached leaves (9-12 per genotype) from plant grown on soil and subjected to dehydration on filter paper at room temperature; WT: wild-type; KO: TDNA insertional knockout line for ; OE8: overexpressing line. (B) Water lost with time from seedlings (30-50 per genotype) grown in vitro and subjected to dehydration at room temperature. Genotypes are as in (A) plus a transgenic line overexpressing a stable form of, harboring the point substitution H91A. 1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 WT KO OE8 H91A 0,2 0, Time (min) Comparative water lost with time