BBX4, a phyB-interacting and modulated regulator, directly interacts with PIF3 to fine tune red light-mediated photomorphogenesis

Yueqin Heng, Yan Jiang, Xianhai Zhao, Hua Zhou, Xuncheng Wang, View ORCID ProfileXing Wang Deng, and Dongqing Xu

PNAS December 17, 2019 116 (51) 26049-26056

Significance

Phytochrome B (phyB) is the predominant red light photoreceptor that transduces red light signals to downstream signaling. On red light exposure, photoactivated phyB interacts with a transcription factor termed PHYTOCHROME INTERACTING FACTOR 3 (PIF3), a repressor of red light signaling, triggering its rapid phosphorylation and subsequent degradation. Thus, phyB-PIF3 defines a critical regulatory hub for red light-mediated seedling development. In this study, we show that B-BOX CONTAINING PROTEIN 4 (BBX4) is a key component involved in the phyB-PIF3 regulatory module. phyB directly interacts with BBX4 and positively controls the abundance of BBX4 in red light. Accumulated BBX4 directly interacts with PIF3 to inhibit its transcriptional activation activity toward target genes, thereby promoting photomorphogenesis.

Abstract

Phytochrome B (phyB) absorbs red light signals and subsequently initiates a set of molecular events in plant cells to promote photomorphogenesis. Here we show that phyB directly interacts with B-BOX CONTAINING PROTEIN 4 (BBX4), a positive regulator of red light signaling, and positively controls its abundance in red light. BBX4 associates with PHYTOCHROME INTERACTING FACTOR 3 (PIF3) and represses PIF3 transcriptional activation activity and PIF3-controlled gene expression. The degradation of BBX4 in darkness is dependent on CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) and the 26S proteasome system. Collectively, BBX4 acts as a key component of the phyB-PIF3–mediated signaling module and fine tunes the red light action. phyB promotes the accumulation of BBX4, which in turn serves to repress PIF3 action through direct physical interaction to promote photomorphogenic development in red light.

https://www.pnas.org/content/116/51/26049

Figure 1:

phyB genetically and physically interacts with BBX4. (A and B) Hypocotyl phenotype (A) and length (B) of 4-d-old Col, bbx4-1, phyB-9, and bbx4-1 phyB-9 seedlings grown in R (115.8 μmol/m²/s) light. The unit of hypocotyl length is millimeters. The experiments were performed 3 times with similar results. The graphs depict one 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 and D) Hypocotyl phenotype (C) and length (D) of 4-d-old Col, YFP-BBX4 #6, phyB-9, and YFP-BBX4 phyB-9 #6 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. (E) Yeast two-hybrid interactions between the BBX4 and phyB. (F) Semi-in vivo pull-down assay of BBX4 with phyB. Total plant protein was extracted from 4-d-old phyB-myc transgenic seedlings grown in R light (115.8 μmol/m²/s). Equal amounts of MBP and MBP-BBX4 proteins were added to total plant protein extracts. The asterisk indicates MBP-BBX4. Actin served as a negative control. (G) BBX4 and phyB colocalize to the nuclear bodies in tobacco cells. CFP-BBX4 and YFP-phyB were transiently coexpressed in tobacco leaves. CFP-GST and YFP-GST served as negative controls. (Scale bars: 5 µm.) (H) BiFC assay showing the interaction of BBX4 with phyB in R light. BBX4 and phyB were fused to the N- and C-terminal fragments of YFP (YFP^N and YFP^C, respectively). Unfused YFP^N and YFP^C fragments served as negative controls. (Scale bars: 20 μm.)