Phytochrome B Positively Regulates Red Light-Mediated ER Stress Response in Arabidopsis

Abstract

Light plays a crucial role in plant growth and development, and light signaling is integrated with various stress responses to adapt to different environmental changes. During this process, excessive protein synthesis overwhelms the protein-folding ability of the endoplasmic reticulum (ER), causing ER stress. Although crosstalk between light signaling and ER stress response has been reported in plants, the molecular mechanisms underlying this crosstalk are poorly understood. Here, we demonstrate that the photoreceptor phytochrome B (phyB) induces the expression of ER luminal protein chaperones as well as that of unfolded protein response (UPR) genes. The phyB-5 mutant was less sensitive to tunicamycin (TM)-induced ER stress than were the wild-type plants, whereas phyB-overexpressing plants displayed a more sensitive phenotype under white light conditions. ER stress response genes (BiP2 and BiP3), UPR-related bZIP transcription factors (bZIP17, bZIP28, and bZIP60), and programmed cell death (PCD)-associated genes (OXI1, NRP1, and MC8) were upregulated in phyB-overexpressing plants, but not in phyB-5, under ER stress conditions. The ER stress-sensitive phenotype of phyB-5 under red light conditions was eliminated with a reduction in photo-equilibrium by far-red light and darkness. The N-terminal domain of phyB is essential for signal transduction of the ER stress response in the nucleus, which is similar to light signaling. Taken together, our results suggest that phyB integrates light signaling with the UPR to relieve ER stress and maintain proper plant growth.

Keywords: UPR signaling pathway; light signaling; phytochrome B; plant ER stress; red light-mediated plant growth.

Figure 1

PhyB is required for endoplasmic reticulum (ER) stress rsponse. (A) Phenotype analysis of 10-day-old seedlings of Wild-type (Ler), phyB-5, and PBG (phyB-GFP overexpression) treated with tunicamycin (TM) grown under continuous white light conditions. (B) Relative fresh weight of shoot parts shown in (A). Three biological replicates were averaged and statistically significant differences compared with WT (Ler) are indicated by asterisks (mean ± SEM; n = 3). (C,D) Transcript levels of BiP2, and BiP3 were quantified using qRT-PCR. The 10-day-old seedlings of Wild-type (Ler), phyb-5, and PBG grown in normal media were treated with TM (500 ng/ml) by vacuum-infiltration. Three biological replicates were averaged and statistically significant differences compared with WT (Ler) are indicated by asterisks (mean ± SEM; n = 3). Asterisks indicate statistically significant differences (Student’s t-test, ^**p < 0.01; and ^*p < 0.05).

Figure 2

PhyB positively regulates unfolded protein response (UPR) signaling. (A–D) Transcript levels of UPR-related genes; bZIP17, bZIP28, bZIP6OS, and bZIP6OU were quantified using qRT-PCR. The 10-day-old seedlings of Wild-type (Ler), phyb-5, and PBG grown in normal media were treated with TM (500 ng/ml) by vacuum-infiltration. Three biological replicates were averaged and statistically significant differences compared with WT (Ler) are indicated by asterisks (mean ± SEM; n = 3). Asterisks indicate statistically significant differences (Student’s t-test, ^***p < 0.001; ^**p < 0.01; and ^*p < 0.05).

Figure 3

PhyB promotes programmed cell death (PCD) through ER, oxidative and light-induced stress responses under ER stress conditions. (A–C) Transcript levels of PCD-related genes; NRP1, MC8, and OXI-1 were quantified using qRT-PCR. The 10-day-old seedlings of Wild-type (Ler), phyb-5, and PBG (phyB-GFP overexpression) grown in normal media were treated with TM (500 ng/ml) by vacuum-infiltration. Three biological replicates were averaged and statistically significant differences compared with WT (Ler) are indicated by asterisks (mean ± SEM; n = 3). Asterisks indicate statistically significant differences (Student’s t-test, ^***p < 0.001; ^**p < 0.01; and ^*p < 0.05).

Figure 4

PhyB Pfr form functions in ER stress relief. (A) Phenotype analysis of Wild-type (Ler), phyb-5, and PBG (phyB-GFP overexpression) treated with tunicamycin (TM) according to light conditions. The 10-day-old seedlings of Wild-type (Ler), phyb-5, and PBG (phyB-GFP overexpression) grown under normal condition were transferred to TM-containing media and the Indicated light conditions; red (1 μmol m⁻² s⁻¹), far-red (1 μmol m⁻² s⁻¹), red: far-red (1:1, total 2 μmol m⁻² s⁻¹), and dark for 3 days. (B) Relative chlorophyll content changes shown in (A). Three biological replicates were averaged and statistically significant differences compared with WT (Ler) are indicated by asterisks (mean ± SEM; n = 3). Asterisks indicate statistically significant differences (Student’s t-test, ^***p < 0.001; ^**p < 0.01; and ^*p < 0.05).

Figure 5

N-terminal of phyB participates in ER stress signal transduction in nucleus. (A) Phenotype analysis of 15-day-old plants treated with TM (10 ng/ml) grown under continuous white light conditions. (B) Relative fresh weight of shoot parts shown in (A). Three biological replicates were averaged and statistically significant differences compared with WT (Ler) are indicated by asterisks (mean ± SEM; n = 3). (C,D) Transcript levels of BiP3, and CNX1 were quantified using qRT-PCR. The 10-day-old seedlings grown in normal media were treated with TM (500 ng/ml) by vacuum-infiltration. Three biological replicates were averaged and statistically significant differences compared with WT (Ler) are indicated by asterisks (mean ± SEM; n = 3). Asterisks Indicate statistically significant differences (Student’s t-test, ^***p < 0.01; ^**p < 0.01; ^*p < 0.05). (E) H₂O₂ accumulation detected by DAB staining. Plants were grown in normal media under continuous white light for 20 days.

Figure 6

A model for the role of phyB in the red light-induced ER stress response. Red light-induced phyB Pfr form activates photomorphogenesis. During this process, phyB is required for ER stress relief by inducing an ER stress response via UPR signaling. In prolonged ER stress, reactive oxygen species (ROS) is highly accumulated in the cell and phyB activates ER, oxidative and light-induced stress-mediated PCD for plant survival.