Temperature modulates tissue-specification program to control fruit dehiscence in Brassicaceae

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1 Supplemental information Temperature modulates tissue-specification program to control fruit dehiscence in Brassicaceae Xin-Ran Li 1, Joyita Deb 1, S. Vinod Kumar 1* and Lars Østergaard 1* 1 John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, United Kingdom * Correspondence: Vinod Kumar (vinod.kumar@jic.ac.uk), Lars Østergaard (lars.ostergaard@jic.ac.uk)

2 Supplemental Figure 1. Shattering quantification by RIT in response to temperature. The halftime of pod shatter of C. rubella (A), L. campestre (B), B. napus cv. Topas (C) and B. napus cv. Westar (D) grown at 22 ºC and 27 ºC, corresponding to Figure 1G. All data are from three biological repeats (n=3), 20 siliques or pods each. Error bars, s.d. *P<0.05, **P<0.01 (Student s t-test).

3 Supplemental Figure 2. Gene expression analysis by real-time RT-PCR in Col-0 stage-16 fruits grown at 17 ºC, 22 ºC and 27 ºC. (A) SEM images of the bases of stage-17b fruits in ind-2 plants grown at 22 ºC. Bar, 100 mm. (B) Crystal violet-stained cross sections of stage-17b fruits in ind-2 plants. Scale bar, 10 mm. (C) Quantitative gene expression analysis by real-time RT-PCR. HSP70 is upregulated in

4 the fruits grown at higher temperature, while the expression levels of FRUITFULL (FUL), SHATTERPROOF 1 (SHP1) and SHP2 in the fruits are not significantly different at each temperature. Relative levels of indicated transcripts are normalized to UBQ10. The corresponding expression level at 22 ºC is set as 1.0. All data are from three biological repeats (n=3). Error bars, s.d. *P<0.05, **P<0.01 (Student s t-test). (D) Confocal images of IND::IND:EYFP in Col-0 stage-16 fruits developed at each temperature. Scale bar, 50 mm. (E) Expression of ADPG1::ADPG1:GFP in Col-0 stage-16 fruits grown at different temperatures. The images were obtained from two independent assays. Scale bar, 50 mm.

5 Supplemental Figure 3. Principle of the IND>GR>IND&GUS construct. In the pop/lhgr system (Craft et al., 2005), the expression pattern of the interested gene (IND) is controlled by the promoter (INDpro) that transcribes the transcription factor LhGR. After Dex induction, LhGR is translocated to the nucleus and binds to the target promoter pop6, and then activates the expression of the GUS reporter gene and the gene of interest (IND) simultaneously. The GUS reporter can be used to monitor the level and pattern of IND expression.

6 Supplemental Figure 4. Regulation of H2A.Z occupancy at each temperature. (A) The expression of HSP70 is upregulated in the stage-16 fruits of H2A.Z:GFP plants at higher temperature. Relative levels of indicated transcripts are normalized to UBQ10. The expression level of HSP70 at 22 ºC is set as 1.0. The data are from three biological repeats (n=3). Error bars, s.d. **P<0.01 (Student s t-test). (B-C) ChIP analysis of H2A.Z occupancy at the SHP1 and SHP2 loci in fruits developed at 17 ºC, 22 ºC and 27 ºC. All data are from three independent ChIP experiments. Error bars, s.d. **P<0.01 (Student s t-test).

7 Supplemental Figure 5. pie1-2, hta9hta11 and hta8hta9hta11 mutant fruits display enhanced dehiscent phenotype. (A) SEM images of the bases of stage-17b fruits in pie1-2, hta9hta11 and hta8hta9hta11 plants grown at 22 ºC. Bar, 100 µm. (B) Shattering quantification of Col-0, pie1-2, hta9hta11 and hta8hta9hta11 fruits at 22 ºC. The data are from three biological repeats (n=3), 20 siliques each. Error bars, s.d. *P<0.05, **P<0.01 (Student s t-test).

8 Supplemental Figure 6. Quantitative gene expression analysis by real-time RT-PCR in Col-0 and arp6-1 stage-16 fruits. The expression of HSP70, SHP1 and SHP2 is upregulated in the arp6-1 fruits, while the levels of FUL transcripts are not significantly changed in Col-0 and arp6-1 fruits. Relative levels of indicated transcripts are normalized to UBQ10. The corresponding expression level in Col-0 fruits is set as 1.0. All data are from three biological repeats (n=3). Error bars, s.d. **P<0.01 (Student s t-test).

9 pind-attb1 GGGGACAAGTTTGTACAAAAAAGCAGGCTCCTTATGTTAATAT CACCGT pind-attb2 GGGGACCACTTTGTACAAGAAAGCTGGGTGCCTCTTTTGGGGC TGTGGTTG IND-attB1 GGGGACAAGTTTGTACAAAAAAGCAGGCTATGATGGAGCCTC AGCCTCA IND-attB2 GGGGACCACTTTGTACAAGAAAGCTGGGTTCAGGGTTGGGAGT TGTGGT UBQ10-54-F ACTTTCTCTCAATTCTCTCTACCG UBQ R CCTCGAGGGTGATTGTCTTT IND-qRT-F CGACGAAGCCATACGTTACA IND-qRT-R GGGTTGGGAGTTGTGGTAAT SHP1-qRT-F ATGTACCTGCGAGCAAAGATAG SHP1-qRT-R GCGACTGGTCATGAGAAGATAC SHP2-qPCR-F CATCTTCCCATCCTCACTTCTC SHP2-qPCR-R CTTGCTGCTCTCTGCTACTT FUL-qRT-F GCGCTCCAGAAGAAGGATAAA FUL-qRT-R TGAGGCAGAAGAACTGAAGAAG HSP70-qRT-F AAACTGCCGGTGGTGTTAT HSP70-qRT-R GCTGGTTGTCTGAATAGGTAGAG IND-ChIP-F AGCCTCACCATCTCCTCATG IND-ChIP-R ATTAGGGTTTCGGGAGGTGG HSP70-ChIP-F TCTCTTAAAGCTCACAGACGAA HSP70-ChIP-R CGATAGCTGGACCTTCACCTT SHP1-ChIP-F TCTGTTTTCAGCAAGTAGGGTCT SHP1-ChIP-R TCTTGAAAATATCTCTCTTTCCTTTTG SHP2-ChIP-F GCAGCAGTTAAGTGGTACTGAGA SHP2-ChIP-R GAAGTGAGGATGGGAAGATGA FUL-ChIP-F ATACTTTGGTCATTTCAGGGTTG FUL-ChIP-R CAGCTGAACCCTACCTCTTCC pind-bsai-f TAGGTCTCAGGAGCCTTATGTTAATATCACCGT pind-bsai-r TAGGTCTCACATTATGCTTCTTTTGGGGCTGTGGTTG OCS-F TAGGTCTCAGCTTGTCCTGCTTTAATGAGATATG OCS-R TAGGTCTCAAGCGGTCCTGCTGAGCCTCGAC ADPG1pro-BsaI-F TAGGTCTCAGGAGAATGGTCATTCTTCGCTTTGT ADPG1pro-BsaI-R TAGGTCTCACATTTCGATTTTGATCGGTTTTGC ADPG1-CDS-BsaI-F TAGGTCTCAAATGGCCCGTTGTTGCAGAC ADPG1-CDS-BsaI-R TAGGTCTCACACCAGAGCATTTAGGAGAAACGGTG gind-eyfp-f TAGTTGGAATGGGTTCGAACCTTATGTTAATATCACCGTAG gind-eyfp-r TTATGGAGTTGGGTTCGAACGGGTTGGGAGTTGTG Supplementary Table 1. Primer list