Fig. 5

H₂O₂ enhances the interaction between BZR1 and PIF4 in vitro and in vivo. a DPI treatment had no significant effects on BL-induced nuclear localization of BZR1-YFP. Transgenic Arabidopsis plants expressing BZR1-YFP were grown on ½ MS medium containing 2 µM PPZ or 2 µM PPZ plus 1 µM DPI in the dark for 5 days, and then treated with 100 nM BL or mock solution for 3 h. Scale bar, 20 μm. b Quantification of fluorescent intensities ratio between nuclear and cytoplasmic signals in a. The standard errors calculated from 50 cells for each treatment. c Immunoblot analysis of phosphorylated and dephosphorylated BZR1 in 5-day-old dark-grown pBZR1:BZR1-YFP using anti-YFP antibody. Actin bands showed protein loadings. d–g In vitro pull-down assays showed that H₂O₂ enhanced, but Cys-63 mutation to Ser reduced, the BZR1 binding to PIF4. e, g show the quantification of pull-down assays (normalized to input) in the d and f, respectively. Error bars represented the s.d. of three independent experiments. *p < 0.05, as determined by a Student’s t-test. h, i rBiFC confocal images showed that H₂O₂ enhanced, but mutation of cysteines reduced the interaction between BZR1 and PIF4 in plants. The fluorescent signals of YFP (BiFC) and RFP (reference) were determined along a line drawn on the confocol images using ImageJ software. Error bars, s.d. (n = 50 images). Different letters above the bars indicated statistically significant differences between the samples (two-way ANOVA, p < 0.05). Scale bar, 10 μm. j Co-immunoprecipitation (CoIP) assays showed that H₂O₂ increased the interaction between BZR1 and PIF4 in plants. The Arabidopsis mesophyll protoplast transformed with BZR1-Myc and/or PIF4-YFP were treated with mock solution or 100 μM H₂O₂ for 1 h, then immunoprecipitation was performed using GFP-Trap agarose beads, and the immunoblots were probed using anti-Myc and anti-YFP antibodies