B-BOX DOMAIN PROTEIN28 Negatively Regulates Photomorphogenesis by Repressing the Activity of Transcription Factor HY5 and Undergoes COP1-Mediated Degradation

Abstract

Plants have evolved a delicate molecular system to fine-tune their growth and development in response to dynamically changing light environments. In this study, we found that BBX28, a B-box domain protein, negatively regulates photomorphogenic development in a dose-dependent manner in Arabidopsis thaliana BBX28 interferes with the binding of transcription factor HY5 to the promoters of its target genes through physical interactions, thereby repressing its activity and negatively affecting HY5-regulated gene expression. In darkness, BBX28 associates with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and undergoes COP1-mediated degradation via the 26S proteasome system. Collectively, these results demonstrate that BBX28 acts as a key factor in the COP1-HY5 regulatory hub by maintaining proper HY5 activity to ensure normal photomorphogenic development in plants.

Figure 1.

bbx28 Is Hypersensitive to Light. Hypocotyl length and phenotype in 4-d-old Col and four independent bbx28 single mutant seedlings grown in darkness ([A] and [B]), W (18.1 μmol/m²/s) ([C] and [D]), B (3.88 μmol/m²/s) ([E] and [F]), R (75.1 μmol/m²/s) ([G] and [H]), and FR (2.05 μmol/m²/s) ([I] and [J]) light conditions. Hypocotyl length is expressed in millimeters. Data are means ± se; n ≥ 20. Letters above the bars indicate significant differences (P < 0.05), as determined by one-way ANOVA with Tukey’s post-hoc analysis. The experiments were performed three times with similar results. The graphs depict the results of one of three experiments.

Figure 2.

BBX28 Transgenic Seedlings Are Hyposensitive to Light. (A) BBX28 transcript levels in Col and various BBX28 transgenic seedlings grown in W light for 4 d, as determined by RT-qPCR. Error bars represent sd of three technical replicates. (B) myc-BBX28 and YFP-BBX28 protein levels in each of three myc- and YFP-tagged BBX28 transgenic seedlings grown in W for 4 d, as determined by immunoblot analysis . (C) to (J) Hypocotyl length and phenotype in 4-d-old Col and six independent BBX28 transgenic lines grown in W (33.3 μmol/m²/s) ([C] and [D]), B (3.28 μmol/m²/s) ([E] and [F]), R (75.1 μmol/m²/s) ([G] and [H]), and FR (2.05 μmol/m²/s) ([I] and [J]) conditions. Hypocotyl length is expressed in millimeters. Data are means ± se; n ≥ 20. In (A), (D), (F), (H), and (J), letters above the bars indicate significant differences (P < 0.05), as determined by one-way ANOVA with Tukey’s post-hoc analysis. The experiments were performed three times with similar results. The graphs depict the results of one of three experiments.

Figure 3.

Hypocotyl Phenotype and Length in hy5-215 bbx28-1 Seedlings Grown in the Light. Hypocotyl phenotype and length in 4-d-old Col, bbx28-1, hy5-215, and hy5-215 bbx28-1 seedlings grown in W (18.1 μmol/m²/s) ([A] and [B]), B (4.49 μmol/m2/s) ([C] and [D]), R (75.1 μmol/m²/s) ([E] and [F]), and FR (2.05 μmol/m²/s) ([G] and [H]) conditions. Hypocotyl length is expressed in millimeters. Data are means ± se; n ≥ 20. Letters above the bars indicate significant differences (P < 0.05), as determined by one-way ANOVA with Tukey’s post-hoc analysis. The experiments were performed three times with similar results. The graphs depict the results of one of three experiments.

Figure 4.

BBX28 Physically Interacts with HY5. (A) Schematic diagram of various constructs used in the yeast two-hybrid assays. Numbers indicate the amino acid positions in HY5 or BBX28. (B) Yeast two-hybrid interactions between the indicated BBX28 and HY5 proteins. (C) Firefly LCI assay showing that BBX28 interacts with HY5 in N. benthamiana leaf cells. The BBX28-LUCⁿ and HY5-LUC^c constructs were transiently coinfiltrated into wild tobacco leaves, and luminescence intensity was detected using LB985 NightSHADE. LUCⁿ and LUC^c served as negative controls. The color scale shows the range of luminescence intensity. Error bars represent sd of three replicates. (D) BiFC assay showing the interaction of BBX28 with HY5. Full-length BBX28 and HY5 were fused to the split N- or C-terminal (YFPⁿ or YFP^c) fragments of YFP. Unfused YFP N-terminal (YFPⁿ), YFP C-terminal (YFP^c), and GST-YFPⁿ fragments were used as negative controls. Merge, merged images of YFP channel and bright field. Bar = 40 μm. (E) Co-IP analysis showing that myc-BBX28 interacts with HA-HY5. Total protein was extracted from wild tobacco leaves transiently expressing 35S:myc-BBX28 alone or together with UBQ10:HA-HY5. The immunoprecipitates were detected using anti-HA and anti-myc antibodies.

Figure 5.

BBX28 Represses the Transcriptional Activation Activity of HY5. (A) to (C) EMSA showing that the presence of increasing amounts of TF-BBX28 decreases the binding of HY5-His to the promoters of FHY1 (A), CHS (B), and HY5 (C). “−” indicates the absence of corresponding of probes or proteins. For HY5-His, “+” indicates that 5.4 pmol is present; for His-TF, “+” and “++” indicate that 2.1 and 4.2 pmol are present, respectively; for His-TF-BBX28, “+” and “++” indicate that 1.4 and 2.8 pmol are present, respectively. TF, trigger factor, a prokaryotic ribosome-associated chaperone protein. FP indicates free probe. (D) and (E) Yeast-one hybrid assays showing that BBX28 inhibits the activation of proFHY1:LacZ (D) and proCHS:LacZ (E) by HY5. Error bars represent sd of four independent yeast cultures. Asterisks represent statistically significant differences (***P < 0.001), as determined by Student’s t test. The experiments were performed three times with similar results. (F) Schematic representation of various constructs used in the transient transfection assay in Arabidopsis protoplasts. Arrow after the 35S promoter indicates the transcriptional start site. −994, −1000, and −752 indicate the length of the BBX22, CHS, and HY5 promoter sequence that was fused to the firefly luciferase gene to create the reporter construct, respectively. (G) to (I) Bar graphs showing that BBX28 represses the activation of the proBBX22:LUC (G), proCHS:LUC (H), and proHY5:LUC (I) reporters by HY5. Error bars represent sd of three independent transient transfections in Arabidopsis protoplasts. Asterisks represent statistically significant differences (***P < 0.001), as determined by Student’s t test. The experiments were performed three times with similar results.

Figure 6.

BBX28 Genetically and Physically Interacts with COP1. (A) and (B) Hypocotyl phenotype (A) and length (B) in 4-d-old Col, bbx28-4, YFP-BBX28 Col #18, cop1-6, bbx28-4 cop1-6, and YFP-BBX28 cop1-6 #18 seedlings grown in darkness. Hypocotyl length is expressed in millimeters. Data are means ± se; n ≥ 20. Letters above the bars indicate significant differences (P < 0.05), as determined by one-way ANOVA with Tukey’s post-hoc analysis. The experiments were performed three times with similar results. The graphs depict the results of one of three experiments. (C) Schematic diagram of the domain structure of COP1 and the truncated COP1 proteins. Numbers indicate the amino acid positions in COP1. (D) Yeast two-hybrid interactions between the indicated COP1 protein and BBX28. (E) BiFC assay showing the interaction of BBX28 with COP1. Full-length BBX28 and COP1 were fused to the split N- or C-terminal (YFPⁿ or YFP^c) fragments of YFP. Unfused YFPⁿ, YFP^c, and GST-YFPⁿ fragments were used as negative controls. Merge, merged images of YFP channel and bright field. Bar = 80 μm. (F) Co-IP analysis showing that myc-BBX28 interacts with COP1 in Arabidopsis seedlings. Four-day-old W light-grown Col and myc-BBX28 Col #44 seedlings were transferred to darkness for 48 h and subjected to a co-IP assay using anti-myc antibodies, with the immunoprecipitates were detected using anti-COP1 and anti-myc antibodies, respectively. Actin served as a negative control.

Figure 7.

BBX28 Undergoes COP1-Mediated Degradation in Darkness. (A) BBX28 transcript levels in 4-d-old Col grown in various light conditions (D, W, B, R, and FR), as determined by RT-qPCR. Error bars represent sd of three technical replicates. Asterisks represent statistically significant differences (***P < 0.01), as determined by Student’s t test. (B) myc-BBX28 protein levels in 4-d-old myc-BBX28 Col #44 transgenic seedlings grown in various light conditions (D, W, B, R, and FR), as determined by immunoblot analysis. Col served as a negative control. (C) YFP-BBX28 protein levels in 4-d-old dark-grown YFP-BBX28 Col #10 transgenic seedlings treated with various concentrations (0, 50, and 100 μm) of MG132. Col treated with DMSO served as a negative control. (D) YFP-BBX28 protein levels in YFP-BBX28 Col #18 and YFP-BBX28 cop1-6 #18 grown in darkness for 4 d. cop1-6 served as a negative control. In (B) to (D), actin was used as a loading control.

Figure 8.

A Proposed Working Model Depicting How the COP1-BBX28-HY5 Regulatory Module Mediates Light Signaling. In darkness, COP1 mediates the degradation of BBX28 and HY5 via the 26S proteasome system to promote skotomorphogenesis. Upon light illumination, COP1 activity is inhibited in a light-dependent manner, allowing BBX28 and HY5 to accumulate. HY5 regulates the expression of numerous downstream target genes to promote photomorphogenesis. BBX28 interacts with HY5 to interfere with its binding to target sites and repress its transcriptional activity, which in turn negatively regulates photomorphogenesis. u, ubiquitin.