Supplemental Data. Steiner et al. Plant Cell. (2012) /tpc

Transcription

1 Supplemental Figure 1. SPY does not interact with free GST. Invitro pull-down assay using E. coli-expressed MBP-SPY and GST, GST-TCP14 and GST-TCP15. MBP-SPY was used as bait and incubated with equal amount of GST, GST-TCP14 or GST-TCP15. Amylose resin was used to pull down MBP-SPY and its interacting proteins. Left panels (pull-down): an anti-gst antibody was used to detect GST, GST-TCP14 or GST-TCP15. The blot was striped and re-probed with anti-mbp antibody to detect MBP-SPY. Right panels (input): amounts of proteins added to the assays. Full-length GST- TCP14 = 80.1 kd, GST-TCP15 = 62.5 kd, MBP-SPY = kd and GST =

2 Supplemental Figure 2. Overexpression of TCP14 under the regulation of the CUC2 or FIL promoters only slightly affected plant development. Three weeks old WT (Ler) and transactivated transgenic plants overexpressing TCP14 under the regulation of CUC2 (CUC2 pro >>TCP14) or FIL (FIL pro >>TCP14) promoters. Bars = 1cm 2

3 Supplemental Figure 3. TCP14 activity requires functional SPY. A. Inflorescences and flowers of WT Ler, transactivated AS1 pro >>TCP14 in Ler, spy-4, transctivated AS1 pro >>TCP14 in spy- 4 and progeny of transctivated AS1 pro >>TCP14 spy-4 plants crossed to WT Ler (spy-4 AS1 pro >>TCP14 x WT). B and C. The activity of the AS1 promoter in the transactivated AS1 pro >>TCP14/GFP WT (AS1 pro >>GFP) and spy-4 (spy-4 AS1 pro >>GFP) seedlings (B) and flowers (C) was confirmed by the detection of the GFP signal using fluorescence binocular (excitation 488 nm, emission 520 nm; Leica MZFL III, St. Gallen, Switzerland). Bars in A - inflorescence = 1 cm; single flower = 1 mm. 3

4 Supplemental Figure 4. Overexpression of TCP14 and TCP15 suppresses petal development. Single flowers of WT Ler and transgenic Ler expressing TCP14 or TCP15 under the regulation of the AS1 promoter in transactivated lines (AS1 pro >>TCP14 and AS1 pro >>TCP15). Bars = 1 mm. 4

5 Supplemental Figure 5. SEC interacts with TCP14 and TCP15. In-vitro pull-down assay using E. coli-expressed MBP-SEC (fulllength SEC), GST, GST-TCP14 and GST-TCP15. MBP-SEC was used as bait and incubated with equal amount of GST, GST-TCP14 or GST-TCP15. Amylose resin was used to pull down MBP-SEC and its interacting proteins. Left panels (pull-down): An anti-gst antibody was used to detect the GST, GST-TCP14 or GST-TCP15. The blot was striped and re-probed with anti-mbp antibody to detect MBP-SPY. Right panels (input): amounts of proteins added to the assays. Full-length GST-TCP14 = 80.1 kd, GST-TCP15 = 62.5 kd, MBP-SEC = kd and GST =

6 Supplemental Figure 6. SEC does not modify GST. ARR5-GST was co-expressed with SEC in E. coli. Duplicate blots were probed to detect GlcNAc modified proteins (GalT) or GST (α-gst). Although the fusion protein was highly expressed it was not modified. The only GlcNAc modified proteins detected were SEC and truncated forms of SEC. 6

7 Supplemental Figure 7. SEC modifies TCP 2, 8, 19, 23 and 24. Clones of TCP coding regions constructed in the vector puni51 were obtained from the ABRC and recombined with phb3-his6 (ABRC) to create a plasmid expressing a HIS-tagged version of the TCP. The HIS-tagged TCP 2, 8, 19, 23 and 24 and an Arabidopsis protein that is not modified by SEC, At2G33430 (C), were coexpressed in E. coli without SEC (-) or with wild-type SEC (+), expressed from pacyc-mal-sec (Scott et al. 2006). Blots containing total E. coli proteins were prepared and proteins bearing GlcNAc modifications were labeled with 3 H-galactose. The expected location of the HIS-TCP protein is indicated by the red dot. Other SEC-modified proteins are truncated forms of SEC or truncated forms of the TCP. TCP 4, 9, 11, and 21 were also tested and either were poorly expressed (TCP4) or had such weak signal that we cannot be confident that they are modified. 7

8 Supplemental Figure 8. The double mutant tcp14 tcp15 and spy-4 share similar phenotypes. A. Rosette leaves (seventh leaf) of WT Col, tcp15, tcp14, tcp14 tcp15 and spy-4 taken from plants grown under short-day conditions (8 h of light). B. Flower buds of WT Col, tcp15, tcp14, tcp14 tcp15 and spy-4. Bar in A = 1 cm, bar in B = 1 mm. 8

9 Supplemental Figure 9. The loss of TCP14 and TCP15 has no effect on GA responses. A. Germination of WT Col, spy-4 and tcp14 tcp15 seeds on 2 mg/l paclobutrazol. B. Number of leaves to flowering in Wt Col, spy-4 and tcp14 tcp15 grown under short day conditions (8 h light). Values are means of ten plants ± SE. 9

10 Supplemental Figure 10. Expression of ARR5, but not TCP14 or TCP15, is promoted by CK. Seedlings were treated with 10 µm BA or water (Mock) and 4 h later RNA was extracted and analyzed by qrt-pcr for the expression of TCP14, TCP15 and ARR5. Values are means of three biological replicates ± SE. 10

11 Supplemental Figure 11. Reducing CK levels suppresses TCP14 overexpression effects. A. Three weeks old WT Ler, AS1 pro >>TCP14, AS1 pro >>CKX3 and AS1 pro >>TCP14/CKX3 plants. B. Inflorescences of WT and the different transgenic lines. C. Representative fifth leaf from mature WT Ler, AS1 pro >>TCP14, AS1 pro >>CKX3 and AS1 pro >>TCP14/CKX3 plants. D. PCR analyses to DNA extracted from WT Ler and the different transgenic lines using primers specific to OP:CKX3 or OP:TCP14. Bar in A and B = 1 cm. 11

12 Supplemental Figure 12. Overexpressing CKX3 suppresses TCP14 activity and this is reversed by exogenous CK. Inflorescences of WT Ler, AS1 pro >>TCP14, AS1 pro >>CKX3 and AS1 pro >>TCP14/CKX3 plants non-treated (left) and treated (right) with 50 µm BA. 12

13 Purpose Primer Sequence (5' to 3' ) Cloning- TCP14F GGCATATGCAAAAGCCAACATCAAG Yeast Two Hybrid TCP14R GGCTCGAGCTAATCTTGCTGATCCTCCT Cloning- Yeast Two Hybrid TCP15F TCP15R AACATATGGATCCGGATCCGGATCA AACTCGAGCTAGGAATGATGACTGGTGC Cloning- invitro pull TCP14_F GGGACAAGTTTGTACAAAAAAGCAGGCTTC ATGCAAAAGCCAACATCAAGT down TCP14_R GGGGACCACTTTGTACAAGAAAGCTGGGTC TAATCTTGCTGATCCTCCT Cloning- invitro pull TCP15_F GGGGACAAGTTTGTACAAAAAAGCAGGCTT CATGGATCCGGATCCGGATCATAACCATCG down TCP15_R GGGGACCACTTTGTACAAGAAAGCTGGGTC TAGGAATGATGACTGGTGC Cloning- invitro pull SEC_F GGGGACAAGTTTGTACAAAAAAGCAGGCTT CATGATCTCGTCCAAAAACGGAGCTGCC down SEC_R GGGGACCACTTTGTACAAGAAAGCTGGGTT TATCTGTCATGTGGGAATTCTAGG Cloning- invitro pull SPY_F GGGGACAAGTTTGTACAAAAAAGCAGGCTA TATGGTGGGACTGGAAGATGATACT down SPY_R GGGGACCACTTTGTACAAGAAAGCTGGGTT CTAGCTAGTGGAGTCCATTCTCTTT PCR analysis TCP14_F ATAATCCAACAAAGCAAGAA OCS_R ATAGGCGTCTCGCATATCTC PCR analysis CKX3_F GGTCAAACGACATCGTGTCA OCS_R ATAGGCGTCTCGCATATCTC PCR analysis TUB_F AGATTCTTCACATCCAGGGTGGTC qrt-pcr analysis qrt-pcr analysis qrt-pcr analysis TUB_R TCP14-RT_F TCP14-RT_R TCP15-RT_F TCP15-RT_R ARR5-RT_F ARR5-RT_R CTCACTACATCGCCTGAACATCTC CGTCGTCTTTGTTTCCTGGT TCTCCTTCTTGCTTTGTTGGA CCTTTGGCTTCTGGTTATGG TTGTTATGGTTCCCCGTCTC GAAGTTCATCGAGCGGTTACTC TTAATCTTCAGATCCTCAAATCCA qrt-pcr TUB-RT_F AAACTCACTACCCCCAGCTTT 13

14 analysis TUB-RT_R GAGAGGAGCAAAACCAACCA Cloning- O- GlcNAc modification Cloning- O- GlcNAc modification Cloning- O- GlcNAc modification Cloning- CUC2 promoter SEC-B1_F SEC-B2_R-1 TCP14_F Gateway TCP14_R Gateway TCP15_F Gateway TCP15_R Gateway CUC2pro_F CUC2pro_R GGGGACAAGTTTGTACAAAAAAGCAGGCTC GATGATCTCGTCCAAAAACGGAGCTG GGGGACCACTTTGTACAAGAAAGCTGGGTT TTATCTGTCATGTGGGAATTCTAGG GGGGACAAGTTTGTACAAAAAAGCAGGCTT CATGCAAAAGCCAACATCAAGT GGGGACCACTTTGTACAAGAAAGCTGGGTC TAATCTTGCTGATCCTCCT GGGGACAAGTTTGTACAAAAAAGCAGGCTT CATGGATCCGGATCCGGATCATAACCATCG GGGGACCACTTTGTACAAGAAAGCTGGGTC TAGGAA TGATGACTGGTGC CTCGAGAGTGAAGACGCGAACAAGTTGC GGATCCTAAGAAGAAAGATCTAAAGCTTTT GTTTGAGA Supplemental Table 1. Primers used in this study. 14