Fig. 3.
BBX4 genetically and physically interacts with PIF3. (A and B) Hypocotyl phenotype (A) and length (B) of 4-d-old Col, bbx4-1, pif3-1 and bbx4-1 pif3-1 seedlings grown in R light (115.8 μmol/m2/s). The unit of hypocotyl length is millimeters. The experiments were performed 3 times, with similar results. The graphs depict 1 of these experiments. Error bars represent SE (n ≥ 20). Letters above the bars indicate significant differences (P < 0.05), as determined by 1-way ANOVA with Tukey’s post hoc analysis. (C) Yeast two-hybrid interactions between the BBX4 and PIF3. (D) FRET between CFP-PIF3 and YFP-BBX4 analyzed by acceptor bleaching in nuclei. (Top) Representative prebleach nuclei coexpressing YFP-BBX4 and CFP-PIF3 excited with a 514-nm or 405-nm laser, resulting in emission from YFP or CFP, respectively. (Bottom) The same nuclei after bleaching excited with a 514-nm or 405-nm laser. (E) The relative intensities of both YFP and CFP inside the nuclei were measured once before and twice after the bleaching, as indicated in D. (F) BiFC assay showing the interaction of BBX4 with PIF3 in red light. BBX4 and PIF3 were fused to the N- and C-terminal fragments of YFP (YFPN and YFPC, respectively). Unfused YFPN and YFPC fragments served as negative controls. (Scale bars: 40 μm.)