BIC 1 acts as a transcriptional coactivator to promote brassinosteroid signaling and plant growth
2020; Springer Nature; Volume: 40; Issue: 1 Linguagem: Inglês
10.15252/embj.2020104615
ISSN1460-2075
AutoresZongju Yang, Baiqiang Yan, Huixue Dong, Guanhua He, Yun Zhou, Jiaqiang Sun,
Tópico(s)Plant tissue culture and regeneration
ResumoArticle14 October 2020free access Source DataTransparent process BIC1 acts as a transcriptional coactivator to promote brassinosteroid signaling and plant growth Zongju Yang Zongju Yang National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China Search for more papers by this author Baiqiang Yan Baiqiang Yan National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China Search for more papers by this author Huixue Dong Huixue Dong National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China Search for more papers by this author Guanhua He Guanhua He National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China Search for more papers by this author Yun Zhou Yun Zhou State Key Laboratory of Crop Stress Adaptation and Improvement, School of Life Sciences, Henan University, Kaifeng, China Search for more papers by this author Jiaqiang Sun Corresponding Author Jiaqiang Sun [email protected] orcid.org/0000-0002-3448-6956 National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China Search for more papers by this author Zongju Yang Zongju Yang National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China Search for more papers by this author Baiqiang Yan Baiqiang Yan National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China Search for more papers by this author Huixue Dong Huixue Dong National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China Search for more papers by this author Guanhua He Guanhua He National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China Search for more papers by this author Yun Zhou Yun Zhou State Key Laboratory of Crop Stress Adaptation and Improvement, School of Life Sciences, Henan University, Kaifeng, China Search for more papers by this author Jiaqiang Sun Corresponding Author Jiaqiang Sun [email protected] orcid.org/0000-0002-3448-6956 National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China Search for more papers by this author Author Information Zongju Yang1,†, Baiqiang Yan1,†, Huixue Dong1, Guanhua He1, Yun Zhou2 and Jiaqiang Sun *,1 1National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China 2State Key Laboratory of Crop Stress Adaptation and Improvement, School of Life Sciences, Henan University, Kaifeng, China † These authors contributed equally to this work *Corresponding author. Tel: +86 15810855809; E-mail: [email protected] The EMBO Journal (2021)40:e104615https://doi.org/10.15252/embj.2020104615 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The BRASSINAZOLE-RESISTANT 1 (BZR1) transcription factor family plays an essential role in plant brassinosteroid (BR) signaling, but the signaling mechanism through which BZR1 and its homologs cooperate with certain coactivators to facilitate transcription of target genes remains incompletely understood. In this study, we used an efficient protein interaction screening system to identify blue-light inhibitor of cryptochromes 1 (BIC1) as a new BZR1-interacting protein in Arabidopsis thaliana. We show that BIC1 positively regulates BR signaling and acts as a transcriptional coactivator for BZR1-dependent activation of BR-responsive genes. Simultaneously, BIC1 interacts with the transcription factor PIF4 to synergistically and interdependently activate expression of downstream genes including PIF4 itself, and to promote plant growth. Chromatin immunoprecipitation assays demonstrate that BIC1 and BZR1/PIF4 interdependently associate with the promoters of common target genes. In addition, we show that the interaction between BIC1 and BZR1 is evolutionally conserved in the model monocot plant Triticum aestivum (bread wheat). Together, our results reveal mechanistic details of BR signaling mediated by a transcriptional activation module BIC1/BZR1/PIF4 and thus provide new insights into the molecular mechanisms underlying the integration of BR and light signaling in plants. Synopsis BZR1 and PIF4 are core transcription factors in brassinosteroid (BR) and light signaling, respectively. Here, Blue-light Inhibitor of Cryptochromes 1 (BIC1) is found as a common coactivator linking BZR1 and PIF4 to synergistically and interdependently activate transcription of common target genes and promote cell elongation. BIC1 positively regulates BR responses. BIC1 interacts with BZR1 and PIF4 to synergistically co-activate transcription of target genes, including PIF4 itself. BIC1 and BZR1/PIF4 interdependently associate with the promoters of common target genes. The interaction between BIC1 and BZR1 is evolutionally conserved in monocot and dicot plants. Introduction Brassinosteroids (BRs) are a group of growth-promoting steroid hormones that control a wide range of plant growth and developmental processes including photomorphogenesis, cell elongation, and flowering (Clouse & Sasse, 1998; Bai et al, 2012; Zhang et al, 2013; Chaiwanon et al, 2016; Choi & Oh, 2016). A lot of BR-insensitive and BR-deficient mutants have been identified by genetic and biochemical analysis (Clouse et al, 1996; Nolan et al, 2019). Generally, BR-deficient mutants display dwarfism and dark-green color phenotype when grown in light, and de-etiolation phenotype when grown in darkness. Through genetic dissection of these mutants, the BR signal transduction cascade from the cell surface receptor kinase BRASSINOSTEROID-INSENSITIVE 1 (BRI1) to the BRASSINAZOLE-RESISTANT 1 (BZR1) family transcription factors has been well clarified (Wang et al, 2012b; Chaiwanon et al, 2016). BRs bind to the receptor kinase complex, including BRI1 and BRI1-ASSOCIATED PROTEIN KINASE 1 (BAK1) (Tang et al, 2010; Wang et al, 2012b; Chaiwanon et al, 2016). BRI1 in turn phosphorylates plasma membrane-anchored cytoplasmic kinases, BRASSINOSTEROID-SIGNALING KINASE 1 (BSK1) and CONSTITUTIVE DIFFERENTIAL GROWTH 1 (CDG1) which phosphorylate BRI1-SUPPRESSOR 1 (BSU1), leading to dephosphorylation and inactivation of the glycogen synthase kinase 3 (GSK3)-like kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2) (Kim et al, 2009; Wang et al, 2012b). In the absence of BR, BIN2 is active and phosphorylates BZR1 and BZR2 (also named BES1 for BRI1-EMS-SUPPRESSOR 1), which lose their DNA-binding activity and remain in the cytoplasm due to binding to 14-3-3 proteins (Kim & Wang, 2010). When BR levels are high, BIN2 is inactive, BZR1 and BES1 are dephosphorylated by protein phosphatase 2A (PP2A) and subsequently translocated to the nucleus to control BR-responsive gene expression (Yin et al, 2002, 2005). BZR1 and BES1 act as the core transcription factors to activate or repress thousands of different target genes’ expression (Vert & Chory, 2006; Sun et al, 2010). In recent years, several BZR1/BES1-interacting proteins have been identified. PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), a bHLH transcription factor, is a member of the family of PHYTOCHROME-INTERACTING FACTORs (PIFs) that directly interact with light-activated phytochromes and regulate various light responses (Choi & Oh, 2016). Identification of genome-wide BZR1 and PIF4 binding sites shows that the two transcription factors share thousands of target genes (Oh et al, 2012). Furthermore, BZR1 interacts with PIF4 to cooperatively regulate the expression of co-target genes and plant growth (Oh et al, 2012). BZR1 also interacts with other light signaling-related factors. For example, BZR1 interacts with LONG HYPOCOTYL 5 (HY5) which in turn attenuates BZR1's transcriptional activity in regulating its target genes related to cotyledon opening (Li & He, 2016). UV light receptor UVR8 physically interacts with BES1-interacting Myc-like 1 (BIM1) and BES1, and represses their DNA-binding activities (Liang et al, 2018). Moreover, the light receptor phytochrome B (phyB) and cryptochromes CRY1/2 interact with BZR1/BES1 to inhibit their DNA-binding activity, respectively (Wang et al, 2018; Dong et al, 2019; He et al, 2019; Wu et al, 2019). Interestingly, our previous study also showed that blue light-activated CRY1 promotes BZR1 phosphorylation and consequently prevents its nuclear localization (He et al, 2019). Taken together, these observations implicate that BZR1 and BES1 are the main integration hubs of light and BR signaling in regulation of hypocotyl elongation. In addition, the gibberellin signaling repressors DELLAs directly interact with BZR1 and inhibit its DNA-binding activity (Bai et al, 2012) and chromatin-remodeling factor PICKLE (PKL) interacts with BZR1 to facilitate the expression of cell elongation-related genes (Zhang et al, 2014). A very recent report showed that salinity stimulates BZR1 deSUMOylation via ULP1a SUMO protease to integrate environmental cues to shape plant growth (Srivastava et al, 2020). Although some BZR1-interacting proteins have been identified, the transcriptional coactivators for BZR1 still remain unknown. In this study, we identify blue-light inhibitor of cryptochromes 1 (BIC1) as a novel BZR1-interacting protein. We showed that BIC1 functions as a transcriptional coactivator for BZR1 to promote BR signaling. Simultaneously, BIC1 also interacts with PIF4 to synergistically and interdependently promote hypocotyl elongation and gene expression. Furthermore, we showed that BIC1 and BZR1/PIF4 associate with the promoters of common target genes interdependently. In addition, we showed that TaBIC1 also interacts with TaBZR1 in bread wheat. Together, we uncover a transcriptional activation module BIC1-BZR1-PIF4 for the activation of target genes to integrate light and BR signaling to coordinate plant growth. Results BIC1 was identified as a BZR1-interacting protein The transcription factor BZR1 in BR signaling pathway plays a vital role in the regulation of cell elongation. To further explore the BZR1-mediated transcriptional regulatory mechanism in the regulation of hypocotyl elongation, we used firefly luciferase (LUC) complementation imaging (LCI) assays to screen for new BZR1-interacting proteins in Nicotiana benthamiana leaves. To this end, sets of genes related to cell elongation were chosen for the LCI assays. BZR1 was firstly fused to the amino-terminal part of LUC (nLUC) to produce nLUC-BZR1; the cell elongation-related proteins including CCA1 (Wang & Tobin, 1998), TOC1 (Mas et al, 2003), ELF3 (Zagotta et al, 1996), LUX (Hazen et al, 2005), and BIC1 (Wang et al, 2016) were fused to the carboxyl-terminal part of LUC (cLUC) to generate corresponding constructs for the LCI assays. The results showed that BZR1 strongly interacted with BIC1 and also interacted with ELF3 and LUX among the tested proteins (Fig 1A). In this study, we focused on the physical and functional interaction of BIC1 and BZR1. To further test the physical interaction between BIC1 and BZR1, we generated the cLUC-BZR1 and nLUC-BIC1 constructs for the LCI assays. The results also demonstrated the physical interaction between BIC1 and BZR1 (Appendix Fig S1A). To further determine the interaction between BIC1 and BZR1, three different cell and biochemical approaches were used. Firstly, we performed Bimolecular Fluorescence Complementation (BiFC) assays in Nicotiana benthamiana leaves. Co-expression of nYFP-BZR1 and cYFP-BIC1 led fluorescence signals mainly in the nucleus, suggesting that BIC1 interacts with BZR1 in plant cell nucleus (Fig 1B). The BiFC assays using the nYFP-BIC1 and cYFP-BZR1 constructs also demonstrated the physical interaction between BIC1 and BZR1 (Appendix Fig S1B). Next, pull-down assays showed that glutathione-S-transferase (GST)-BIC1 fusion proteins were pulled down by maltose-binding protein (MBP)-BZR1, but not MBP alone (Fig 1C), suggesting that BIC1 interacts with BZR1 in vitro. To perform co-immunoprecipitation (Co-IP) assays, the 35S:BZR1-MYC/35S:BIC1-YFP double transgenic plant was generated by genetic crossing. Co-IP assays showed that yellow fluorescent protein (YFP)-tagged BIC1 (BIC1-YFP) but not YFP was immunoprecipitated by BZR1-MYC (Fig 1D, Appendix Fig S1C), demonstrating that BIC1 interacts with BZR1 in vivo. In addition, our LCI assays showed that the BIC1 homolog BIC2 also interacts with BZR1 (Appendix Fig S2). Together, these results suggest that BIC1 (and also BIC2) interacts with BZR1 in vitro and in vivo. Figure 1. BIC1 was identified as a BZR1-interacting protein Luciferase complementation imaging (LCI) assays showing that BIC1 interacts with BZR1. nLUC-BZR1 was co-transformed with cLUC-CCA1, cLUC-TOC1, cLUC-ELF3, cLUC-LUX, cLUC-BIC1, cLUC, respectively, in leaf epidermal cells of Nicotiana benthamiana as indicated. BiFC assays showing the interaction of BIC1 and BZR1 mainly in nucleus. Leaf epidermal cells of Nicotiana benthamiana were co-transformed with nYFP-BZR1 and cYFP-BIC1. BF, bright field. Scale bars represent 20 μm. In vitro pull-down assays showing BIC1 directly interacts with BZR1. Purified GST-BIC1 proteins were incubated with MBP or MBP-BZR1 for the MBP pull-down assay. Arrowhead indicates specific bands. Co-immunoprecipitation (Co-IP) assays showing the interaction between BIC1 and BZR1 in vivo. Seedlings were grown for 6 days under long-day conditions. The immunoprecipitates were detected using anti-GFP and anti-MYC antibodies, respectively. * indicates specific bands. The cross-reacting lower band on IP blot is the heavy chain after IP. Source data are available online for this figure. Source Data for Figure 1 [embj2020104615-sup-0002-SDataFig1.zip] Download figure Download PowerPoint To map the interaction domains of BZR1 and BIC1, we divided the full-length proteins of BZR1 and BIC1 into the N-terminal and C-terminal parts, respectively. The N-terminal and C-terminal parts of BZR1 were fused with cLUC to generate cLUC-BZR1-NT and cLUC-BZR1-CT, respectively. LCI assays showed that strong interaction signals were observed in the samples co-expressing nLUC-BIC1/cLUC-BZR1-CT, indicating that the C-terminal region of BZR1 mainly mediates the interaction with BIC1 in plant cells (Appendix Fig S3A and B). On the other hand, the N-terminal and C-terminal parts of BIC1 were fused with nLUC to generate nLUC-BIC1-NT and nLUC-BIC1-CT, respectively. LCI assays showed that the C-terminal part of BIC1, containing a conserved CID domain (CRY-interacting domain) (Wang et al, 2016), interacts with BZR1 in plant cells (Appendix Fig S3C and D). BIC1 positively regulates BR responses BIC1, which was initially identified as a repressor of flowering via inhibiting CRY2 phosphorylation, promotes hypocotyl elongation (Wang et al, 2016, 2017), but the underlying mechanism remains unknown. In order to clarify the physiological significance of the interaction between BIC1 and BZR1, we assessed the sensitivity of Col-0, 35S:BIC1-YFP, 35S:BIC1-Flag, and bic1bic2 double mutant plants to gradually increasing concentrations of epibrassinolide (eBL, an active form of brassinosteroids) under white light, dark, red light, and blue light, respectively. The results showed that the 35S:BIC1-YFP and 35S:BIC1-Flag transgenic plants were significantly hypersensitive to exogenous BR treatment in hypocotyl elongation compared with Col-0 under white light and blue light conditions but not in dark and red light, whereas the bic1bic2 double mutants were insensitive to exogenous BR treatment under white light and blue light conditions (Fig 2A and B, Appendix Fig S4A–F). Next, we analyzed the BR-induced expression patterns of several BR-responsive genes, including PRE1, PRE5, IAA19, and SAUR-AC (Yin et al, 2002, 2005; Yu et al, 2011; Oh et al, 2012, 2014a) in the Col-0 and bic1bic2 seedlings. Quantitative RT–PCR (RT–qPCR) showed that these genes were strongly upregulated by eBL treatment in Col-0, whereas the BR induction was largely abolished in the bic1bic2 double mutants (Fig 2C). Together, these results indicate that BICs positively regulate BR responses. Figure 2. BIC1 positively regulates BR responses Hypocotyl elongation phenotypes of the BIC1 overexpression and mutant lines in response to BR treatment. Col-0, 35S:BIC1-YFP, 35S:BIC1-Flag and bic1bic2 seedlings were grown for 6 days on 1/2 MS medium supplemented with 1 μM brassinazole (BRZ) plus different concentrations of epibrassinolide (eBL). Images of the representative seedlings are shown in (A), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (B). Data are means ± SD; n > 20. Scale bars, 2 mm. RT–qPCR analysis of BZR1-activated gene expression in the bic1bic2 double mutants. The 6-day-old Col-0 and bic1bic2 seedlings grown on 1/2 MS medium supplemented with 1 μM BRZ were treated with 5 μM eBL (+eBL) or not (−eBL) for 1 h. The PP2A gene served as internal control. Data are means ± SD (n = 3). Heat map of BIC1-regulated genes. The Col-0 and 35S:BIC1-YFP seedlings were grown on 1/2 MS medium for 10 days. Three biologic replicates were performed. The colored bar beneath the map indicates fold change (log2 value). BIC1 facilitates the expression of cell elongation genes. RT–qPCR analysis of genes selected in transcriptomic data. The Col-0, 35S:BIC1-YFP and 35S:BIC1-Flag seedlings were grown for 5 days under long-day conditions. The PP2A gene served as internal control. Data are means ± SD (n = 3). Gene Ontology analyses of BIC1-regulated genes. Numbers indicate -log P Value. The Venn diagram shows significant overlap between BIC1-regulated and BZR1-target and PIF4-target genes. Distribution of BIC1 activated or repressed genes among the overlap genes in (G). Download figure Download PowerPoint To comprehensively analyze differentially expressed genes between Col-0 and BIC1-overexpression plants, we conducted RNA-sequencing (RNA-seq) experiments. Transcriptomic data showed that sets of genes including BR-activated and cell elongation-related genes were upregulated in the 35S:BIC1-YFP transgenic seedlings compared with Col-0 (Fig 2D). We then verified the transcriptomic data using RT–qPCR. The results showed that the transcript levels of BZR1 target genes and other cell elongation-related genes were all upregulated in the 35S:BIC1-YFP and 35S:BIC1-Flag transgenic seedlings, whereas the expression levels of the “florigen” gene FLOWING LOCUS T (FT) were significantly down-regulated (Fig 2E), consistent with the delayed flowering phenotype of BIC1-overexpression plants (Wang et al, 2016). Gene Ontology (GO) enrichment analysis indicated that BIC1-regulated genes are implicated in light response and hormone responses such as auxin and brassinosteroid (Fig 2F). On the other hand, BIC1-regulated genes were also enriched for GO terms associated with transcription regulator activity (Fig 2F). We further analyzed the overlaps between BIC1-regulated genes and BZR1/PIF4 target genes (Sun et al, 2010; Oh et al, 2012) and found that 20.8% (320/1537) of BIC1-regulated genes are BZR1 targets and 24.7% (379/1537) of BIC1-regulated genes are PIF4 targets (Fig 2G). Among the 162 co-regulated genes by BIC1, BZR1, and PIF4 (Fig 2G), most of these genes (79%, 128/162) are BIC1-activated genes at the transcriptional level (Fig 2H), suggesting that BIC1 might act as a transcriptional coactivator to facilitate the BZR1/PIF4-mediated activation of common target genes. BIC1 genetically interacts with BZR1 To further investigate the genetic relationship between BIC1 and BZR1, we analyzed the hypocotyl elongation phenotypes of the single transgenic plants 35S:BZR1-MYC and 35S:BIC1-YFP as well as the double transgenic plants 35S:BZR1-MYC/35S:BIC1-YFP. We showed that the transcript and protein levels of BIC1 were comparable in the 35S:BIC1-YFP and 35S:BZR1-MYC/35S:BIC1-YFP genetic background (Appendix Fig S5A and B), suggesting that overexpression of BZR1 alone does not affect the transcript and protein abundance of BIC1. Notably, the transcript and phosphorylation status of BZR1 were similar in the 35S:BZR1-MYC and 35S:BZR1-MYC/35S:BIC1-YFP genetic background (Appendix Fig S5C and D), revealing that overexpression of BIC1 does not affect the transcriptional expression and phosphorylation status of BZR1. Phenotypic analyses showed that the double transgenic plants 35S:BZR1-MYC/35S:BIC1-YFP exhibited longer hypocotyl than that of its parent plants (Fig 3A and B). We further generated the pBIC1:BIC1 transgenic plants under the control of its native promoter and pBIC1:BIC1/35S:BZR1-MYC double transgenic plants by genetic crossing. As expected, the double transgenic plants pBIC1:BIC1/35S:BZR1-MYC exhibited longer hypocotyl than that of its parent plants (Appendix Fig S6A and B). These results well demonstrated that BIC1 and BZR1 synergistically promote hypocotyl elongation. Considering that BZR1-overexpression lines are hypersensitive to exogenous BR treatment in the promotion of hypocotyl elongation (Oh et al, 2014b), we further examined the genetic interaction of BIC1 and BZR1 in BR-induced hypocotyl elongation. The results showed that the hypocotyls of 35S:BIC1-YFP and 35S:BZR1-MYC were significantly elongated after eBL treatment compared with Col-0 (Fig 3C and D). Notably, the 35S:BZR1-MYC/35S:BIC1-YFP double transgenic plants exhibited remarkably longer hypocotyl than that of its parent plants (Fig 3C and D), indicating that BIC1 and BZR1 synergistically promote BR response. The BR-induced hypocotyl elongation assays using gradually increasing concentrations of eBL well demonstrated the synergistic relationship between BIC1 and BZR1 in mediating BR response (Fig 3E). Figure 3. BIC1 and BZR1 synergistically promote hypocotyl elongation BIC1 and BZR1 synergistically promote hypocotyl elongation. The Col-0, 35S:BZR1-MYC, 35S:BIC1-YFP and 35S:BZR1-MYC/35S:BIC1-YFP plants were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (A), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (B). Data are means ± SD (n > 20). **P < 0.01, as determined by Student's t-test. Scale bar, 2 mm. BR signaling is enhanced in the 35S:BZR1-MYC/35S:BIC1-YFP plants. Seedlings were grown for 6 days on medium supplemented with 1 μM brassinazole (BRZ) plus a gradient of concentrations of epibrassinolide (eBL) under long-day conditions. Images of the representative seedlings when grown with 500 nM eBL (+eBL) or not (−eBL) are shown in (C), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (D) and (E). Data are means ± SD (n > 20). **P < 0.01, as determined by Student's t-test. Scale bar, 2 mm. BZR1-mediated hypocotyl elongation phenotype is dependent on the function of BIC1 and BIC2. Seedlings were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (F), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (G). Data are means ± SD (n > 20). **P < 0.01, n.s. indicates no significant difference (Student's t-test). Scale bar, 2 mm. BZR1-mediated hypocotyl elongation phenotype is dependent on the function of BIC1 and BIC2. The Col-0, 35S:BZR1-MYC, bic1bic2, 35S:BZR1-MYC/bic1bic2 seedlings were grown on medium supplemented with 1 μM BRZ plus different concentrations of eBL for 6 days and then the hypocotyl lengths were measured. Data are means ± SD (n > 20). Download figure Download PowerPoint It has been known that the bzr1-1D gain-of-function mutant plants display shorter hypocotyls than wild type in light (Wang et al, 2002; He et al, 2005) (Appendix Fig S7A and B). However, the bzr1-1D mutation showed a promotional effect on hypocotyl elongation in the BIC1-overexpression background under light (Appendix Fig S7A and B), suggesting that BZR1's function of promoting cell elongation requires BIC1. To further investigate the genetic relationship between BIC1 and BZR1, we introduced the 35S:BZR1-MYC transgene into the bic1bic2 double mutants background by genetic crossing. The results showed that both the BZR1-overexpression induction of hypocotyl elongation and the BZR1-enhanced BR response in promoting hypocotyl elongation were dependent on the function of BIC1 and BIC2 (Fig 3F–H). BIC1 has transcriptional activation activity Although we demonstrate that BIC1 and BZR interact physically and genetically, the underlying mechanism remains unclear. It has been reported that BIC1 acts as an inhibitor of CRY2 function through suppressing the blue light-dependent dimerization, phosphorylation, and degradation of CRY2 (Wang et al, 2016, 2017). Moreover, the phosphorylation status of BZR1 plays a critical role in the function of BZR1 (He et al, 2002; Li & Nam, 2002; Yang & Wang, 2017). However, the phosphorylation status of BZR1 was not affected by overexpression of BIC1 (Appendix Fig S5D). On the other hand, we also tested whether BR treatment affects the transcriptional expression and protein stability of BIC1. The results showed that the transcriptional expression pattern and protein levels of BIC1 were not obviously altered by eBL or brassinazole (BRZ, a BR biosynthesis inhibitor) treatments under normal conditions (Appendix Fig S8A and B). We further investigated the effect of eBL on the BIC1 protein stability under the protein synthesis inhibitor cycloheximide (CHX) treatment. As shown in Fig 4A, the BIC1 protein abundance gradually decreased after CHX treatment, whereas the decreasing of BIC1 abundance was largely blocked by eBL treatment. These results indicate that BR treatment can stabilize the BIC1 protein. Figure 4. BR-stabilized BIC1 has transcriptional activation activity BR enhances the stability of BIC1 proteins. The 5-day-old 35S:BIC1-YFP transgenic plants were treated with 100 μM cycloheximide (CHX) or co-treated with 100 μM CHX and 1 μM epibrassinolide (eBL). Samples were collected at indicated time points, and BIC1-YFP protein levels were analyzed by Western blots using anti-GFP antibody. Actin was used to verify equal protein loadings. Intensity of the bands was measured using Adobe Photoshop CS3 Extended program. Three independent experiments were performed with similar results. Scheme represents full-length and truncated versions of BIC1. NT, amino-terminal domain; CT, carboxyl-terminal domain; CID, CRY-interacting domain. Transactivation analysis of BIC1 using a yeast assays. The GAL4 DNA-binding domain (BD) alone was used as the negative control. SD-W, synthetic dextrose medium lacking Trp; SD-WH, synthetic dextrose medium lacking both Trp and His. Constructs used for BIC1 transcriptional activity assays as shown in (E). VP16 was used as a positive control. TATA, TATA box for DNA binding; LUC, firefly luciferase; REN, Renilla luciferase; NOS, nopaline synthase terminator. Transient transcriptional activity analysis in Arabidopsis protoplasts illustrating the transcriptional activation activity of BIC1. The relative luciferase activities were calculated by normalizing the LUC values against REN. Error bars represent SD (n = 3). **P < 0.01, as determined by Student's t-test. Constructs used for BIC1 transcriptional activity assay as shown in (G). Transient expression assays in Arabidopsis protoplasts showing that BIC1 and BZR1 synergistically activate PRE5 promoter. The ProPRE5:LUC reporter was co-transformed with the indicated effector constructs. The LUC/REN ratio represents the ProPRE5:LUC activity relative to the internal control (REN driven by the 35S promoter). Error bars represent SD (n = 3). BIC1-mediated hypocotyl elongation was reduced by the addition of SRDX motif. Seedlings were grown for 5 days under long-day conditions. Images of the representative seedlings are shown in (H), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (I). #1, #2, #3 represent different 35S:BIC1-SRDX-Flag transgenic lines. Data are means ± SD (n > 20). **P < 0.01, as determined by Student's t-test. Scale bar, 2 mm. Source data are available online for this figure. Source Data for Figure 4 [embj2020104615-sup-0003-SDataFig4.zip] Download figure Download PowerPoint Considering that BIC1 directly interacts with the transcription factor BZR1 as shown in this study, we wondered whether BIC1 has transcriptional activity. To test this idea, we first performed transactivation activity assays in yeast. The full-length BIC1 was fused in-frame with the GAL4 DNA-binding domain in the pGBKT7 vector, and the resulting construct was transformed into the Gold yeast strain. The growth of yeast carrying pGBKT7-BIC1 on selective medium (SD-W-H) indicated that BIC1 protein has transcriptional activation activity compared to the empty pGBKT7 vector used as the negative control (Fig 4B and C). Furthermore, based on a pre
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