Arabidopsis MSBP1 Is Activated by HY5 and HYH and Is Involved in Photomorphogenesis and Brassinosteroid Sensitivity Regulation  Shi Qiu-Ming , Yang Xi , Song Li , Xue Hong-Wei   Molecular Plant  Volume 4, Issue 6, Pages 1092-1104 (November 2011) DOI: 10.1093/mp/ssr049 Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 1 MSBP1 Expression Is Up-Regulated and Induced by Different Types of Light. (A) Quantitative real-time RT–PCR analyses indicated that MSBP1 expression was up-regulated under various types of light, particularly under far-red, blue, and white light. One-week-old Arabidopsis seedlings were grown under different light conditions (continuous blue light, red light, far-red light, and white light with 16-h light/8-h dark cycles, with same conditions for the control, 1C), and hypocotyls were isolated and used for analysis. MSBP1 expression in the dark was set at 1.0, and the relative gene expression is presented as the average ± the standard deviation (SD) from three independent biological experiments. The data were statistically analyzed using a two-tailed Student’s t-test (** P < 0.01, compared with plants grown in darkness). (B) MSBP1 promoter–reporter gene (GUS) fusion studies confirmed that MSBP1 expression was induced by various light conditions, particularly in the hypocotyls. One-week-old Arabidopsis seedlings grown under different light conditions were used for observation. (C) Quantitative real-time RT–PCR analyses showed that MSBP1 expression was induced by white light. The seedlings were grown in the dark after germination for 7 d and then transferred to light for 1, 2, 4, or 6 h. Hypocotyls were isolated and used for analysis. MSBP1 expression under darkness was set at 1.0, and the relative gene expression is presented as the average ± standard deviation (SD) from three independent repeats. The data were statistically analyzed using a two-tailed Student’s t-test (** P < 0.01, compared with the MSBP1 expression under darkness). (D) Enhanced (O-MSBP1) or suppressed (A-MSBP1) expression of MSBP1 affected the hypocotyl length under various light conditions. The seedlings were grown under various light conditions for 6 d and the hypocotyl lengths were measured. The data were statistically analyzed using a two-tailed Student’s t-test (* P < 0.05; ** P < 0.01, compared with WT plants under the corresponding type of light) and are presented as the average ± standard error (SE) (n > 20). Molecular Plant 2011 4, 1092-1104DOI: (10.1093/mp/ssr049) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 2 Suppressed Expression of MSBP1 Shows Insensitivity to the Fluence-Rate Response. Suppressed MSBP1 expression (A-MSBP1) resulted in longer hypocotyl length and insensitivity to fluence-rate response under far-red, blue, and white light, but not red light. Enhanced expression of MSBP1 (O-MSBP1) resulted in shorter hypocotyls under all light conditions, but did not affect fluence-rate response. R, red light; FR, far-red light; B, blue light; W, white light. Seedlings were grown under different light conditions for 6 d and the hypocotyl lengths were measured (left panel). The relative length (the length under each lowest light intensity was set at 1.0) was calculated and is presented as the average ± SE (n > 30). The experiment was repeated, with similar results. Statistical analysis was performed with a two-tailed Student’s t-test (* P < 0.05; ** P < 0.01, compared with WT plants). Molecular Plant 2011 4, 1092-1104DOI: (10.1093/mp/ssr049) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 3 MSBP1 Expression Is Altered under COP1, HY5, or HYH Deficiency. (A) Analysis of Arabidopsis seedlings expressing HY5:mGFP5 driven by the CaMV35S promoter showed a similar HY5 expression pattern to MSBP1. Green fluorescence was observed throughout the seedlings grown under light (left), but was concentrated in the cotyledons and the joint between the cotyledons and hypocotyls in the dark-grown plants (middle). The upper and lower parts of the hypocotyls from the dark-grown plants are shown in the right panel. The seedlings were grown in darkness or light (16-h light/8-h dark) for 6 d. (B) Western-blot analysis showed the differential accumulation of HY5 protein in plants grown under various types of light. (C) Quantitative real-time RT–PCR analyses showed that the MSBP1 transcript is down-regulated in the hypocotyls of hy5, hyh, or hy5 hyh mutants grown under light (left panel) and is elevated in the cop1 mutant when grown in darkness (right panel). Seedlings grown in darkness (cop1) or under light (16-h light /8-h dark, hy5, hyh, or hy5 hyh) for 6 d after germination were used for analysis. Hypocotyls were isolated and used for analysis. The MSBP1 transcript of the hy5 hyh mutant (left panel) or WT plant (right panel) was set as 1.0, and the relative expression is presented as the average ± SD from three independent repeats. The data were statistically analyzed using a two-tailed Student’s t-test (** P < 0.01, compared with the MSBP1 transcript of WT plants). Molecular Plant 2011 4, 1092-1104DOI: (10.1093/mp/ssr049) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 4 HY5 and HYH Bind to the MSBP1 Promoter Region. (A) Schematic diagram of the HY5-binding region of the MSBP1 promoter. The promoter region of MSBP1 (starting from –1 500 bp) was divided into four parts (MPP1, 2, 3, and 4), and each part was cloned into the pG221 vector and co-transformed with PPC86 constructs containing HY5 or HYH coding regions (upper panel). A detailed analysis of the MPP2 region is shown in the bottom panel. The position of the GATA-box is highlighted (black box). (B) Yeast one-hybrid analysis revealed that yeast cells expressing HY5 or HYH proteins show β-galactosidase activity when the DNA fragment of the MPP2 region is co-transformed. Yeast cells containing the MPP2 region and mock vector PPC86 were used as negative controls. (C) The electrophoretic mobility shift assay (EMSA) confirmed the binding of HY5 and HYH with the promoter region of MSBP1. Free probes are indicated with stars and shift bands are indicated with arrows. NC, negative control. (D) Quantitative real-time PCR analysis by Chromatin IP (CHIP) showed the significant enrichment of the MSBP1 promoter in transgenic lines expressing p35S:HY5–GFP or p35S:HYH–GFP. About 1/10 of total DNA without immunoprecipitation was used as the positive control (PC), and a sample immunoprecipitated without the GFP antibody served as the negative control (NC). The DNA region of the MSBP1 promoter (–142 to –45) not containing the GATA-box served as a negative control for fragment level (MPNC). The amount of each fragment in the positive control was set as 1.0, and the relative fragment amount is presented as the average ± SD from three independent repeats. The data were statistically analyzed using a two-tailed Student’s t-test (** P < 0.01, compared with NC). Experiments were biologically repeated (R1 or R2). Molecular Plant 2011 4, 1092-1104DOI: (10.1093/mp/ssr049) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 5 The GATA-Box Is Essential for the Binding Specificity of HY5 and HYH. (A) The sequence of the MSBP1 promoter region containing the GATA-box and the corresponding mutation or deletion mutants. (B) The electrophoretic mobility shift assay (EMSA) indicated that the mutated DNA region deleting the GATA-box failed to compete with the labeled MPP2n. For the assay, 0.1 μg of HY5 protein or 0.5 μg of HYH protein was used. Unlabeled MPP2n fragments with mutations or deletions of the GATA-box element served as cold competitors for the assays. Normal MPP2n fragments were used as the positive control. All competitions were 12 times the labeled probe used. Free probes are indicated with stars and shift bands are indicated with arrows. Molecular Plant 2011 4, 1092-1104DOI: (10.1093/mp/ssr049) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 6 Altered Expression of MSBP1 Results in Altered BL Response under Different Light Conditions. Enhanced expression of MSBP1 (O-MSBP1) resulted in a hyposensitive response to BL treatment under all light conditions, whereas suppressed MSBP1 expression (A-MSBP1) only displayed hypersensitive responses under far-red, blue, and white light. D, dark; R, red light; FR, far-red light; B, blue light; W, white light. Seedlings were grown under different light conditions for 6 d and the hypocotyl lengths were measured (left panel). The relative length (the length without BL treatment was set at 1.0) was calculated and is presented as the average ± SE (n > 30). The experiment was repeated and similar results were obtained. Statistical analysis was performed with a two-tailed Student’s t-test (* P < 0.05; ** P < 0.01, compared with WT plants). Molecular Plant 2011 4, 1092-1104DOI: (10.1093/mp/ssr049) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 7 hy5 and hy5 hyh Mutants Present Altered Responses to Exogenous BL in Light, but Not Darkness. (A) hy5 or hy5 hyh mutants showed altered responses to BL treatment when grown in light (16-h light/8-h dark). The lengths of hypocotyls from 6-day-old seedlings were measured (left panel) and the relative length was calculated (right panel; the length without BL treatment was regarded as 100%). The data were statistically analyzed using a two-tailed Student’s t-test (* P < 0.05; ** P < 0.01, mutant plants were compared with corresponding WT plants with the same treatment) and are presented as the average ± SE (n > 20). (B) No differences were detected between dark-grown WT plants and hy5 or hy5 hyh mutants that were provided with different concentrations of exogenous BL. Seedlings were grown in darkness for 6 d. Hypocotyl lengths were measured and are presented as the average ± SE (n > 20). Statistical analysis was performed using a two-tailed Student’s t-test (** P < 0.01, mutant plants were compared with corresponding WT plants with the same treatment). (C) Quantitative real-time RT–PCR analyses indicated that the BR biosynthesis-related genes CPD, ROT3, and DWF4 were suppressed in hy5, hyh, and hy5 hyh mutants. One-week-old Arabidopsis mutant seedlings, Wassilewskija (WS), or WS treated with BL (100 nM) for 3 h were used for analysis. The expression of the gene tested in WT samples was set at 1.0 and the relative expression was calculated and presented as the average ± SD from three independent repeats. The data were statistically analyzed using a two-tailed Student’s t-test to confirm significant differences (P < 0.01) compared to WT plants (in all tests). Molecular Plant 2011 4, 1092-1104DOI: (10.1093/mp/ssr049) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 8 Photoreceptor-Related Mutants Show Altered Responses to Exogenous BL under Various Light Conditions. The cry1, cry2, and phyA mutants showed altered responses to BL treatment under blue light (cry1, cry2) and far-red light (phyA), whereas phyB exhibited a similar response under red light. The cop1 mutant shows a light-type reaction to exogenous BL when grown in darkness. Seedlings were grown under various light conditions for 6 d and the hypocotyl lengths were measured (left panel). The relative length (the length without BL treatment was set at 1.0) was calculated and presented as the average ± SE (n > 20). Statistical analysis was performed with a two-tailed Student’s t-test (* P < 0.05; ** P < 0.01, compared with WT plants). Molecular Plant 2011 4, 1092-1104DOI: (10.1093/mp/ssr049) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 9 Hypothetical Model for the Effects of MSBP1 on Photomorphogenesis. In darkness, HY5 and HYH are degraded by COP1, leading to a deficiency of MSBP1 and cell elongation (elongated hypocotyls). With light, HY5 and HYH activate MSBP1 expression through direct binding, which leads to suppression of hypocotyl cell elongation. In addition, BR regulates HY5 accumulation, and MSBP1 suppresses BR signaling by interacting with BAK1 to synergistically regulate the cell elongation of the hypocotyls. Molecular Plant 2011 4, 1092-1104DOI: (10.1093/mp/ssr049) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions