Journal: Plant Physiology

Article Title: ABI5-BINDING PROTEIN2 Coordinates CONSTANS to Delay Flowering by Recruiting the Transcriptional Corepressor TPR2 ¹

doi: 10.1104/pp.18.00865

Figure Lengend Snippet: AFP2 reinforces CO and TPR2 to coordinate flowering time. A, Y3H analysis to detect the formation of the CO-AFP2-TPR2 complex. Yeast cotransformed with these three constructs could grow well on the nonselective medium lacking Leu, Trp, and uracil (−L/−W/−U), but only yeast harboring constructs that had positive interactions were able to grow on restrictive growth medium supplemented with 10 mm 3-aminotriazole plus 2% (w/v) Gal and lacking His/Leu/Trp/Ura (−H/−L/−W/−U). B, In vitro pull-down analysis of the interactions among CO, AFP2, and TPR2. Recombinant GST-TPR2 and MBP-CO proteins were produced in E. coli. After cell lysis, cell extracts of GST-TPR2 and MBP-CO were mixed with HIS-AFP2, HIS-AFP∆E, or HIS-AFP2∆J, respectively, and then incubated with magnetic anti-His-coupled magnetic beads. His-tagged full-length or truncated AFP2 was precipitated and washed using a magnetic stand, eluted by boiling in SDS loading buffer, and separated by SDS-PAGE. GST-TPR2 and MBP-CO were detected by immunoblotting. C, Co-IP analysis of the CO-AFP2-TPR2 complex in vivo. The CO-HA/afp2 transgenic line was crossed with AFP-ox/afp2 to obtain CO-HA/AFP2-ox/afp2, which was subjected to Co-IP analysis. Total proteins were extracted from CO-HA/AFP2-ox/afp2 and immunoprecipitated with anti-Flag agarose beads, and the immunoprecipitated proteins were detected with anti-TPR2 antibody. D, Flowering phenotype of the afp2 mutant and the indicated transgenic lines in the afp2 mutant background. The photos were taken at 18 d after seeds germination. Bar = 3 cm. E, Flowering times based on the total rosette leaf number under LD conditions. Data are means ± sd of three biological replicates. For each line, 20 plants were observed. Bars with different letters are significantly different at P < 0.05 (Tukey’s test). F, RT-qPCR analysis of CO transcript levels in the afp2 mutant and different transgenic lines in the afp2 background. IPP2 was used as an internal control. Data are means ± sd of three biological replicates. Bars with different letters are significantly different at P < 0.05 (Tukey’s test). 3AT, 3-aminotriazole.

Article Snippet: The pulled-down proteins were extensively washed with buffer (50 m m Tris-HCl, pH 7.4, 100 m m NaCl, and 0.6% [v/v] Triton X-100) before the samples were resolved on 8% (w/v) SDS-PAGE gels and analyzed by protein gel blot analysis using anti-MBP (1:5000, New England Biolabs), anti-HIS (1:300, Qiagen), and anti-GST (1:3000, Invitrogen), followed by a mouse secondary antibody (1:5000, Promega) and the ECL system (Invitrogen).

Techniques: Construct, In Vitro, Recombinant, Produced, Lysis, Incubation, Magnetic Beads, SDS Page, Western Blot, Co-Immunoprecipitation Assay, In Vivo, Transgenic Assay, Immunoprecipitation, Mutagenesis, Quantitative RT-PCR