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. 2016 Jul 5;113(27):7655-60.
doi: 10.1073/pnas.1607687113. Epub 2016 Jun 20.

BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation

Dongqing Xu  1 , Yan Jiang  1 , Jigang Li  2 , Fang Lin  1 , Magnus Holm  3 , Xing Wang Deng  4
Affiliations

BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation

Dongqing Xu et al. Proc Natl Acad Sci U S A. .

Abstract

BBX21 (also known as SALT TOLERANCE HOMOLOG 2), a B-box (BBX)-containing protein, has been previously identified as a positive regulator of light signaling; however, the precise role of BBX21 in regulating seedling photomorphogenesis remains largely unclear. In this study, we report that CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) interacts with BBX21 in vivo and is able to ubiquitinate BBX21 in vitro. Thus, BBX21 is targeted for 26S proteasome-mediated degradation in dark-grown Arabidopsis seedlings in a COP1-dependent manner. Moreover, we show that BBX21 binds to the T/G-box in the ELONGATED HYPOCOTYL 5 (HY5) promoter and directly activates HY5 expression in the light. Transgenic seedlings overexpressing BBX21 exhibit dramatically shortened hypocotyls in the light, and this phenotype is dependent on a functional HY5. Taken together, our data suggest a molecular base underlying BBX21-mediated seedling photomorphogenesis, indicating that BBX21 is a pivotal component involved in the COP1-HY5 regulatory hub.

Keywords: Arabidopsis; BBX21; COP1; HY5; photomorphogenesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. S1.
Fig. S1.
Expression pattern of BBX21 during dark-to-light transition. (A) Real-time qPCR analyses of BBX21 transcript levels in Col grown in darkness for 4 d, then transferred to white light (112.3 μmol/m2/s) for 1–48 h. (B) Immunoblot analysis of YFP-BBX21 levels in YFP-BBX21 bbx21-1 (#12) overexpression seedlings grown in the dark for 4 d, then transferred to white light (112.3 μmol/m2/s) for 1–48 h. bbx21-1 served as a negative control, and anti-CSN6 served as a loading control.
Fig. S2.
Fig. S2.
Transcript and protein levels of BBX21 in BBX21 overexpression seedlings. (A) Real-time qPCR analyses of BBX21 transcript levels in Col, bbx21-1, and BBX21 overexpression seedlings grown under white light for 4 d. (B) Immunoblot analysis of myc-BBX21 levels in myc-BBX21 bbx21-1 overexpression seedlings grown in the dark for 4 d. The molecular weight of myc-BBX21 is ∼43 kDa. (C) Immunoblot analysis of YFP-BBX21 levels in YFP-BBX21 bbx21-1 overexpression seedlings grown in the dark for 4 d. The molecular weight of YFP-BBX21 is ∼72 kDa. In B and C, bbx21-1 served as a negative control, and anti-actin served as a loading control.
Fig. 1.
Fig. 1.
BBX21 is controlled by COP1 in dark-grown seedlings. (A) Co-IP analysis showing that BBX21 interacts with COP1 in vivo. Four-day-old white light-grown bbx21-1 and myc-BBX21 bbx21-1 (#17) seedlings were transferred to darkness for 16 h and then subjected to a co-IP assay using anti-myc antibodies, with the immunoprecipitates detected using anti-COP1 and anti-myc antibodies, respectively. Actin served as a negative control. (B) BiFC assay showing the interaction of BBX21 with COP1 in onion epidemal cells. Full-length COP1 and BBX21 were fused to the split N- or C-terminal fragments of YFP (YN-COP1 or YC-BBX21). Nuclear localized CFP-CSU1 served as a marker for successful transfection. Unfused YFP N-terminal (YN) or C-terminal (YC) fragments served as negative controls, as indicated. DAPI staining marked the positions of nuclei. Dic, differential interference contrast in light microscope mode; Merge, merged images of YFP channel, DAPI, and Dic. (Scale bar: 100 μm.) (C) COP1 ubiquitinates BBX21 in vitro. Ubiquitination assays were performed in a reaction mix containing UBE1 (E1), rice 6×His-Rad6 (E2), and myc-tagged ubiquitin (myc-Ub). Ubiquitinated MBP-COP1 and 6×His-TF-BBX21 were detected by anti-ubiquitin and anti-myc monoclonal antibodies, respectively. The “+” and “−” indicate presence and absence, respectively. (D) Immunoblot analysis of myc-BBX21 protein levels in dark-grown myc-BBX21 bbx21-1 (#17) transgenic seedlings treated with DMSO or various concentrations of MG132 (50, 100, or 200 μM) for 3 h. (E) Immunoblot analysis of YFP-BBX21 protein levels in YFP-BBX21 bbx21-1 (#12) and YFP-BBX21 bbx21-1 cop1-4 (#12) transgenic seedlings grown in the dark for 4 d. (F) Immunoblot analysis of myc-BBX21 protein levels in myc-BBX21 bbx21-1 (#17) and myc-BBX21 bbx21-1 cop1-6 (#17) transgenic seedlings grown in the dark for 4 d. In D–F, bbx21-1 served as a negative control, and anti-HSP90 or anti-actin served as a loading control.
Fig. S3.
Fig. S3.
COP1 controls BBX21 abundance in dark-grown seedlings. (A) Hypocotyl cells of dark-grown YFP-BBX21 bbx21-1 (#12) seedlings treated with DMSO or 100 μM MG132 for 3 h analyzed by fluorescence microscopy. (B) Relative YFP fluorescence intensity in hypocotyls of YFP-BBX21 bbx21-1 (#12) transgenic seedlings treated with DMSO or 100 μM MG132 for 3 h. (C) Immunoblot analysis of YFP-BBX21 protein levels in dark-grown YFP-BBX21 bbx21-1 (#12) transgenic seedlings treated with DMSO or 100 μM MG132 for 3 h. bbx21-1 served as a negative control, and anti-actin served as a loading control. (D) Analysis of YFP-BBX21 in hypocotyl with fluorescence microscopy. YFP-BBX21 bbx21-1 (#12) and YFP-BBX21 bbx21 cop1-4 (#12) transgenic seedlings were grown in the dark for 4 d. (E) Relative YFP fluorescence intensity in hypocotyls of YFP-BBX21 bbx21-1 (#12) and YFP-BBX21 bbx21-1 cop1-4 (#12) transgenic seedlings grown in the dark for 4 d. In A and D, the pictures are representative images taken from the hypocotyls. YFP, YFP channel image; Dic, differential interference contrast in light microscope mode; Merge, merged images of YFP and Dic. (Scale bar: 50 μm.) In B and E, data were obtained from two independent experiments. At least 10 seedlings were measured each time. Fluorescence intensity was measured using Image J software.
Fig. 2.
Fig. 2.
BBX21 transgenic seedlings are hypersensitive to light. (A, C, E, and G) Fluence rate response curves of 4-d-old Col, bbx21-1, and BBX21 transgenic seedlings grown under white light (A), blue light (C), red light (E), and far-red light (G) conditions. Hypocotyl lengths are in millimeters. Data are mean ± SE; n ≥20. (B, D, F, and H) Hypocotyl phenotypes of 4-d-old Col, bbx21-1, and BBX21 transgenic seedlings grown under white light (33.3 μmol/m2/s) (B), blue light (6.37 μmol/m2/s) (D), red light (59.5 μmol/m2/s) (F), and far-red light (1.46 μmol/m2/s) (H) conditions. In B, D, F, and H, Col, bbx21-1, and various transgenic seedlings are separated by dotted lines. The experiments were performed three times, with similar results. The graphs depict one of these experiments.
Fig. S4.
Fig. S4.
The hypocotyl lengths of BBX21 overexpression seedlings were indistinguishable from those of the WT in darkness. Hypocotyl phenotypes (A) and lengths (B) of Col, bbx21-1, and BBX21 overexpression seedlings grown in darkness for 4 d. In A, Col, bbx21-1, and various transgenic seedlings are separated by dotted lines.
Fig. S5.
Fig. S5.
BBX21 overexpression seedlings accumulate more anthocyanin. Anthocyanin contents of Col, bbx21-1, and BBX21 overexpression seedlings grown for 4 d under white light (112.3 μmol/m2/s) (A), blue light (0.62 μmol/m2/s) (B), red light (6.78 μmol/m2/s) (C), and far-red light (1.46 μmol/m2/s) (D) conditions. FW, fresh weight. Data are mean ± SE; n = 3. The experiments were performed three times, with similar results.
Fig. 3.
Fig. 3.
BBX21 directly up-regulates the expression of HY5. (A) Real-time qPCR analyses of HY5 transcript levels in Col, bbx21-1, and BBX21 overexpression seedlings grown under white light for 4 d. (B) ChIP assays showing that BBX21 associates with the HY5 promoter in vivo. ChIP was performed with anti-myc monoclonal antibodies, and the ChIP DNA was analyzed by real-time qPCR. Error bars represent SD of three technical replicates. (C) Diagram of the WT and various mutated versions of the HY5 promoter subfragments used in the EMSA assays. The WT T/G-box and E-box elements are shown in red, and nucleotide substitutions in the mutant subfragments are shown in blue and underscored. (D and E) EMSA assays using 6×His-TF-BBX21 and the WT (D) or various mutated versions (E) of the HY5 promoter subfragments as the probes. 6×His-TF protein served as the negative control. The “+” and “−” indicate presence and absence, respectively. FP, free probe.
Fig. S6.
Fig. S6.
BBX21 does not regulate the expression of COP1, HYH, PIF1, and PIF3. Real-time qPCR analyses of COP1 (A), HYH (B), PIF1 (C), and PIF3 (D) transcript levels in Col, bbx21-1, and BBX21 overexpression seedlings grown under white light for 4 d. Error bars represent SD of three technical replicates.
Fig. S7.
Fig. S7.
BBX21 controls the expression of HY5-regulated genes. Real-time qPCR analyses of CHS (A), CHI (B), DFR (C), LDOX, (D) F3H (E), and F3′H (F) transcript levels in Col, bbx21-1, and BBX21 overexpression seedlings grown under white light for 4 d.
Fig. S8.
Fig. S8.
ChIP assays showing that BBX21 is not associated with the HYH promoter in vivo. ChIP was performed with anti-myc antibodies, and ChIP DNA was analyzed by real-time qPCR. Error bars represent SD of three technical replicates.
Fig. 4.
Fig. 4.
Mutation of HY5 restores the hyperphotomorphogenic phenotypes of BBX21 overexpressors in the light. Hypocotyl phenotypes and lengths of 4-d-old Col, various mutants, and transgenic seedlings grown in darkness (A and B), white light (33.3 μmol/m2/s) (C and D), red light (59.5 μmol/m2/s) (E and F), blue light (6.37 μmol/m2/s) (G and H), and far-red light (1.46 μmol/m2/s) (I and J) conditions. Hypocotyl lengths are 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. In A, C, E, G, and I, Col, various mutant and transgenic seedlings are separated by dotted lines. The experiments were performed three times, with similar results. The graphs depict one of these experiments.
Fig. 5.
Fig. 5.
A working model depicting how BBX21 is involved in the COP1-HY5 signaling hub. In darkness, activated COP1 directly targets downstream substrates including BBX21, HY5, and HYH for ubiquitination, which subsequently triggers their proteolysis via the 26S proteasome system. On light illumination, the activity of COP1 is inhibited, resulting in the accumulation of BBX21, HY5, and HYH. Accumulated BBX21, HY5, and HYH directly bind to the T/G-box, and CAM7 associates with the E-box in the HY5 promoter. Together, these factors induce HY5 expression, leading to a high abundance of HY5, which regulates a large number of genes involved in promoting photomorphogenesis. ub, ubiquitination; u, ubiquitin.
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