BIC1 acts as a transcriptional coactivator to promote brassinosteroid signaling and plant growth

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.

BIC1 interacts with BZR1 and PIF4 transcription factors to synergistically activate target gene expression, thereby linking brassinosteroid and light signaling in 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.

1. 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.
2. 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.
3. 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.
4. 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.

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.

• A, B
  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.
• B
  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).
• C
  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).
• D
  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).
• E
  Gene Ontology analyses of BIC1‐regulated genes. Numbers indicate ‐log P Value.
• F
  The Venn diagram shows significant overlap between BIC1‐regulated and BZR1‐target and PIF4‐target genes.
• G
  Distribution of BIC1 activated or repressed genes among the overlap genes in (G).

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.

• A, B
  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.
• C–E
  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.
• F, G
  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.
• D
  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).

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.

• A
  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.
• B
  Scheme represents full‐length and truncated versions of BIC1. NT, amino‐terminal domain; CT, carboxyl‐terminal domain; CID, CRY‐interacting domain.
• C
  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.
• D
  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.
• E
  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.
• F
  Constructs used for BIC1 transcriptional activity assay as shown in (G).
• 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).
• H, I
  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.

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 previous report we divided BIC1 into two parts (Wang et al, 2016): N‐terminal domain and C‐terminal domain containing CID. The yeast assays indicated that the C‐terminal domain but not N‐terminal domain of BIC1 has activation activity (Fig 4B and C). We carried out transient dual‐luciferase reporter system (DLR) (Hellens et al, 2005) in Arabidopsis protoplasts to confirm that BIC1 has transcriptional activation activity. BIC1 CDS was fused with the GAL4 DNA‐binding domain (DB) under the control of 35S promoter. The reporter construct consisted of a 35S promoter with the 5X GAL4 DNA‐binding site driving a Luciferase (LUC) reporter gene (Fig 4D). The Renilla Luciferase (REN) gene under the control of the 35S promoter served as an internal control (Fig 4D). The transcriptional activation motif VP16 was used as a positive control (Fig 4D). These constructs were expressed transiently in Col‐0 protoplasts, and bioluminescence was measured. As shown in Fig 4E, transfection of VP16 effector gene together with the reporter genes resulted in stronger LUC activity than control (Fig 4E). Similarly, LUC activity was enhanced by more than twofold by transient expression of BIC1 (Fig 4E), confirming that BIC1 has transcriptional activation activity. As BIC1 and BZR1 display a synergistic relationship in promoting BR responses and hypocotyl elongation (Fig 3A–E) and since BIC1 has transcriptional activation activity, we hypothesize that BIC1 and BZR1 cooperate to synergistically activate downstream genes. To verify our hypothesis, DLR assays were carried out. We generated a ProPRE5:LUC reporter construct (Fig 4F). Co‐expression of BZR1 or BIC1 alone together with ProPRE5:LUC in Arabidopsis protoplasts led to an increased LUC activity (Fig 4G). When the ProPRE5:LUC reporter was co‐expressed with both BZR1 and BIC1, the activation of LUC activity was extremely enhanced (Fig 4G). These results support our hypothesis that BIC1 acts as a transcriptional coactivator of BZR1 to synergistically activate target genes.

To further investigate the essential role of BIC1 activation activity for its function, we generated independent 35S:BIC1‐SRDX-Flag overexpression lines (SRDX, a repressor domain (Heyl et al, 2008; Hiratsu et al, 2003)). We selected three different 35S:BIC1‐SRDX-Flag lines for further analyses, in which the transcript levels and protein abundance of BIC1 were comparable to those in the 35S:BIC1‐Flag overexpression line (Appendix Fig S9A and B). Phenotypic analyses showed that the hypocotyl lengths of 35S:BIC1‐SRDX-Flag #1, #2, and #3 were significantly shorter than that of the 35S:BIC1‐Flag transgenic plants (Fig 4H and I), suggesting that addition of a transcriptional repressive motif can impair the function of BIC1 in promoting hypocotyl elongation. Meanwhile, the transcript levels of BR‐induced genes in the 35S:BIC1‐SRDX-Flag transgenic lines were notably lower than those of the 35S:BIC1‐Flag transgenic plants (Appendix Fig S9C–E), indicating that the transcriptional activation activity of BIC1 is required for the induction of target genes. It should be mentioned that the 35S:BIC1‐SRDX-Flag/pifQ plants still show longer hypocotyls compared with the wild type, which may be due to the remaining activity of BIC1 or the possibility that the function of BIC1 is not solely dependent on its transcriptional activation activity.

BIC1 and PIF4 synergistically and interdependently promote hypocotyl elongation and activate target genes

Previous studies have shown that BZR1 cooperates with PIF4 to promote hypocotyl elongation (Oh et al, 2012, 2014a; Ibanez et al, 2018). To determine whether BIC1 also interacts with PIF4, we first performed LCI assays to investigate the physical interaction between BIC1 and PIF4. LCI assays showed that strong interaction signals were observed in the samples co‐expressing nLUC‐BIC1 and cLUC‐PIF4 (Fig 5A), indicating that BIC1 indeed interacts with PIF4 in plant cells. Next, pull‐down assays were carried out, and GST‐BIC1 and MBP‐PIF4 recombinant proteins from E. coli were purified to test whether BIC1 directly interacts with PIF4. As shown in Fig 5B, MBP‐PIF4, but not MBP, was able to pull down BIC1, indicating that BIC1 directly interacts with PIF4 in vitro. For Co‐IP assays, the 35S:PIF4‐MYC/35S:BIC1‐YFP double transgenic plants were generated by genetic crossing between 35S:BIC1‐YFP and 35S:PIF4‐MYC transgenic plants. Co‐IP assays showed that BIC1‐YFP was immunoprecipitated by PIF4‐MYC, suggesting that BIC1 interacts with PIF4 in vivo (Fig 5C). In addition, our LCI assays showed that the BIC2 also interacts with PIF4 (Appendix Fig S10). Taken together, different biochemical approaches demonstrated that BIC1 (and also BIC2) interacts with PIF4 in vitro and in vivo.

Figure 5. BIC1 physically and genetically interacts with PIF4.

• A
  LCI assays showing that BIC1 interacts with PIF4. Leaf epidermal cells of Nicotiana benthamiana were co‐transformed with nLUC‐BIC1 or nLUC and cLUC‐PIF4 or cLUC as indicated.
• B
  In vitro pull‐down assays showing that BIC1 directly interacts with PIF4. Purified GST‐BIC1 proteins were incubated with MBP or MBP‐PIF4 for the MBP pull‐down assay. Arrowhead indicates specific bands.
• C
  Co‐IP assays showing the interaction between BIC1 and PIF4 in vivo. The 35S:BIC1‐YFP, 35S:PIF4‐MYC and 35S:PIF4‐MYC/35S:BIC1‐YFP transgenic plants were grown for 6 days under long‐day conditions. The immunoprecipitates were detected using anti‐GFP and anti‐MYC antibodies, respectively.
• D, E
  Phenotypic analyses showing that BIC1 and PIF4 synergistically promote hypocotyl elongation. The Col‐0, 35S:BIC1‐YFP, 35S:PIF4‐MYC and 35S:PIF4‐MYC/35S:BIC1‐YFP plants were grown for 5 days under long‐day conditions. Images of the representative seedlings are shown in (D), and the hypocotyl lengths of the indicated genotypes were measured and are shown in (E). Data are means ± SD (n > 20). **P < 0.01, as determined by Student's t‐test. Scale bar, 2 mm.
• E
  RT–qPCR analysis showing that BIC1 and PIF4 synergistically activate PIF4 target genes. The seedling growth conditions were same as (D). The PP2A gene was used as an internal control. Data are means ± SD (n = 3).
• G, H
  Phenotypic analyses showing that PIF4‐induced hypocotyl elongation is partially dependent on the function of BIC1 and BIC2. The Col‐0, bic1bic2, PIF4‐MYC and PIF4‐MYC/bic1bic2 plants were grown for 5 days under long‐day conditions. Images of the representative seedlings are shown in (G), and the hypocotyl lengths of indicated genotypes were measured and are shown in (H). Data are means ± SD (n > 20). **P < 0.01, as determined by Student's t‐test. Scale bar, 2 mm.
• G
  RT–qPCR analysis showing that PIF4‐induced expression of target genes is partially dependent on the function of BIC1 and BIC2. The seedling growth conditions were same as (G). The PP2A gene was analyzed as an internal control. Data are means ± SD (n = 3). **P < 0.01, as determined by Student's t‐test.
• J, K
  Phenotypic analyses showing that BIC1‐induced hypocotyl elongation is largely dependent on the function of PIFs. The Col‐0, pifQ, 35S:BIC1‐Flag, 35S:BIC1‐flag/pifQ #7 and BIC1‐flag/pifQ #8 plants were grown for 5 days under long‐day conditions. Images of the representative seedlings are shown in (J), and the hypocotyl lengths of indicated genotypes were measured and are shown in (K). 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.

We next evaluated the genetic interaction relationship between BIC1 and PIF4. Phenotypic analyses showed that the hypocotyl length of double transgenic plants 35S:PIF4‐MYC/35S:BIC1‐YFP was significantly longer than that of the single transgenic plants 35S:PIF4‐MYC and 35S:BIC1‐YFP (Fig 5D and E). We also generated the pBIC1:BIC1/35S:PIF4‐MYC double transgenic plants by genetic crossing. As expected, the pBIC1:BIC1/35S:PIF4‐MYC double transgenic plants exhibited longer hypocotyl than that of its parent plants (Appendix Fig S11A and B). These results indicated that BIC1 and PIF4 synergistically promote hypocotyl elongation. Consistently, the expression of PIF4 target genes including YUC8, IAA19, and IAA29 (Oh et al, 2012; Sun et al, 2012, 2013) in the double transgenic plants 35S:PIF4‐MYC/35S:BIC1‐YFP was significantly enhanced compared to that of the single transgenic plants 35S:PIF4‐MYC and 35S:BIC1‐YFP (Fig 5F), suggesting that BIC1 and PIF4 synergistically activate target genes. Western blot analyses confirmed that the protein levels of PIF4 or BIC1 in the single or double transgenic plants are comparable (Appendix Fig S12A and B).

We then generated the 35S:PIF4‐MYC/bic1bic2 plants by introducing the 35S:PIF4‐MYC transgene into the bic1bic2 double mutants background through genetic crossing. Phenotypic analyses showed that the long hypocotyl phenotype of 35S:PIF4‐MYC plant was partially impaired in the bic1bic2 background, suggesting that PIF4‐promoted hypocotyl elongation is partially dependent on the function of BICs (Fig 5G and H). Meanwhile, RT–qPCR showed that the expression of PIF4 target genes (YUC8, IAA19, and IAA29) was obviously reduced in the bic1bic2 background (Fig 5I), indicating that PIF4 induction of target genes was also partially dependent on the function of BICs. RT–qPCR and Western blot analyses confirmed that the expression levels of PIF4 in the 35S:PIF4‐MYC and 35S:PIF4‐MYC/bic1bic2 plants were comparable (Appendix Fig S12C and D). On the other hand, we wondered whether the function of BIC1 is dependent on PIFs. To address this question, we generated different 35S:BIC1‐Flag transgenic lines in the pifQ quadruple mutants background (pifQ, pif1pif3pif4pif5) (Shin et al, 2009). Two independent lines 35S:BIC1-flag/pifQ #7, #8 in which the transcript levels and protein abundance of BIC1 were comparable to those in the 35S:BIC1‐Flag transgenic line were selected for further assays (Appendix Fig S13A and B). As shown in Fig 5J and K, the BIC1‐induced hypocotyl elongation was largely abolished in the pifQ mutants background, indicating that the promotional effect of BIC1 on hypocotyl elongation is dependent on the function of PIFs. Consistently, the BIC1 activation of target genes such as YUC8, IAA19, and IAA29 was also impaired in the pifQ mutant background (Appendix Fig S13C–E), suggesting that BIC1‐triggered transcriptional activation of target genes requires the function of PIFs.

BIC1 and BZR1/PIF4 associate with the promoters of common target genes interdependently

The data presented above indicate that BIC1 serves as a transcriptional coactivator for the transcription factors BZR1 and PIF4. So, we wondered whether BIC1 is also associated with the promoters of BZR1/PIF4 target genes. To test this idea, we performed chromatin immunoprecipitation (ChIP) assays using the 6‐day‐old Col‐0 and 35S:BIC1‐Flag seedlings. The results showed that BIC1 was indeed associated with the promoter regions of BZR1/PIF4 target genes such as SAUR‐AC and IAA19 (Fig 6A and B). We further investigated whether the association of BIC1 with the target genes is dependent on the transcription factors PIFs. The ChIP assays using the 35S:BIC1‐Flag and 35S:BIC1‐Flag/pifQ transgenic plants showed that the association of BIC1 with the target genes was significantly reduced in the pifQ mutant background, demonstrating that the association of BIC1 with the target genes is dependent on PIFs (Fig 6C). On the other hand, we tested whether BICs affect the association of BZR1/PIF4 with the chromatin of their target genes. Interestingly, we found that the binding of BZR1/PIF4 to the promoter of their target genes was significantly reduced in the bic1bic2 double mutants (Fig 6D and E), suggesting that BICs facilitate the accessibility of BZR1/PIF4 to the chromatin of their target genes. Taken together, these results suggest that BIC1 and BZR1/PIF4 associate with the promoters of common target genes interdependently.

Figure 6. BIC1 and BZR1/PIF4 associate with the promoters of common target genes interdependently.

1. Diagram of the promoter structures of SAUR‐AC and IAA19. Arrows indicate the primer positions for ChIP‐qPCR. E‐box, CACATG. G‐box, CACGTG.
2. ChIP‐qPCR analysis showing the association of BIC1 with the promoter regions of BZR1/PIF4 target genes. The 6‐day-old Col‐0 and 35S:BIC1‐Flag seedlings were used for ChIP assays.
3. ChIP‐qPCR analysis showing that the association of BIC1 with the promoter regions of BZR1/PIF4 target genes is dependent on PIFs. The 6‐day-old 35S:BIC1‐Flag and 35S:BIC1‐Flag/pifQ seedlings were used for ChIP assays.
4. ChIP‐qPCR analysis showing that the enrichment of BZR1 in the promoter regions of its target genes is dependent on BICs. The 10‐day-old 35S:BZR1‐MYC and 35S:BZR1‐MYC/bic1bic2 seedlings were treated with 1 μM epibrassinolide (eBL) for 3 h and then collected for ChIP assays.
5. ChIP‐qPCR analysis showing that the enrichment of PIF4 in the promoter regions of its target genes is dependent on BICs. The 6‐day-old 35S:PIF4‐MYC and 35S:PIF4‐MYC/bic1bic2 seedlings were used for ChIP assays.

Data information: For (B to E), chromatin of each sample was immunoprecipitated (IP) using an anti‐Flag, anti‐MYC or not (Mock). Precipitated DNA was quantified by qPCR, and DNA enrichment is displayed as the ratio between IP and Mock, normalized to that of PP2A as an internal control. Data are means ± SD (n = 3).

BIC1 cooperates with BZR1/PIF4 to activate the transcription of PIF4

In the above transcriptomic data (Fig 2D), we noticed that the transcript levels of PIF4 were obviously upregulated in the 35S:BIC1‐YFP transgenic plants compared to those in the wild type (Col‐0) (Fig 2D). In the next step, we performed RT–qPCR to verify this result. In line with our transcriptomic data, the transcriptional expression levels of PIF4 in the 35S:BIC1‐YFP and 35S:BIC1‐Flag transgenic lines were indeed higher than those in the wild type (Fig 7A). Consistently, the expression of pPIF4:GUS was clearly enhanced in the basal region of 35S:BIC1‐YFP hypocotyls compared to that of the wild type (Appendix Fig S14). In contrast, the transcriptional expression levels of PIF4 in the bic1bic2 double mutants were reduced compared to those in the wild type (Fig 7A). These results indicated that BIC1 promotes the transcriptional expression of PIF4. To further evaluate the activation of PIF4 by BIC1, we generated the chemical‐inducible BIC1 overexpression plants (pERGW‐BIC1). As shown in Fig 7B, the transcriptional expression of PIF4 was rapidly upregulated by the induced overexpression of BIC1, indicating that BIC1 might directly regulate the transcription of PIF4. To test whether BIC1 associates with the promoter of PIF4, the ChIP assays were performed using the 6‐day‐old Col‐0 and 35S:BIC1‐Flag seedlings. The results demonstrated that BIC1 was indeed associated with the promoter of PIF4 (Fig 7C), illustrating that BIC1 directly regulates the transcription of PIF4.

Figure 7. BIC1 activates PIF4 expression and interacts with BZR1 in bread wheat.

1. RT–qPCR analysis showing that BIC1 upregulates the transcriptional expression of PIF4. The Col‐0, 35S:BIC1‐YFP, 35S:BIC1‐Flag and bic1bic2 plants were grown for 5 days under long‐day conditions. The PP2A gene served as internal control. Data are means ± SD (n = 3). Different letters indicate significant differences (Fisher's LSD, P < 0.05).
2. Transcript levels of BIC1 and PIF4 in transgenic plants containing a chemical‐inducible construct pERGW‐BIC1. 8‐day-old pERGW‐BIC1 seedlings were treated with 10 μM estradiol for indicated time points before harvest for RNA extraction and RT–qPCR analysis. The PP2A gene served as internal control. Data are means ± SD (n = 3).
3. ChIP‐qPCR analysis showing the association of BIC1 with the promoter regions of PIF4. The top panel shows the diagram of the promoter structure of PIF4. Arrows indicate the primer positions for ChIP‐qPCR. G‐box, CACGTG. The 6‐day-old Col‐0 and 35S:BIC1‐Flag seedlings were used for ChIP assays. The chromatin of each sample was immunoprecipitated (IP) using an anti‐Flag or not (Mock). Precipitated DNA was quantified by qPCR, and DNA enrichment is displayed as the ratio between IP and Mock, normalized to that of PP2A as an internal control. Data are means ± SD (n = 3). *P < 0.05, as determined by Student's t‐test.
4. RT–qPCR analysis showing the transcript levels of endogenous PIF4 in the 5‐day-old Col‐0, 35S‐PIF4 and PIF4‐HA seedlings. The PP2A gene served as internal control. Data are means ± SD (n = 3).
5. Transient expression assays in Arabidopsis protoplasts showing that BIC1 and BZR1/PIF4 synergistically activate PIF4 promoter. The ProPIF4:LUC reporter was co‐transformed with the indicated effector constructs. The LUC/REN ratios represent the ProPIF4:LUC activity relative to the internal control (REN driven by the 35S promoter). Error bars represent SD (n = 3). *P < 0.05, as determined by Student's t‐test.
6. LCI assays showing that TaBIC1 interacts with TaBZR1. Leaf epidermal cells of Nicotiana benthamiana were co‐transformed with nLUC‐TaBIC1 or nLUC and cLUC‐TaBZR1 or cLUC as indicated.
7. In vitro pull‐down assays showing that TaBIC1 directly interacts with TaBZR1. Purified GST‐TaBIC1 proteins were incubated with MBP or MBP‐TaBZR1 for the MBP pull‐down assay. Arrowhead indicates specific band.
8. BiFC assays showing the interaction of TaBIC1 and TaBZR1 in nucleus. Leaf epidermal cells of Nicotiana benthamiana were co‐transformed with nYFP‐TaBIC1 and cYFP‐TaBZR1. The yellow dots represent interaction signals. BF, bright field. Scale bars, 20 μm.
9. A propose working model for a transcriptional regulatory module BIC1‐BZR1-PIF4. BIC1 acts as a transcriptional coactivator and interacts with the transcription factors BZR1 and PIF4 to activate PIF4 and other target genes, consequently promoting cell elongation.

Source data are available online for this figure.

Consistent with a recent report demonstrating that PIF4 is able to bind to the promoter of PIF4 itself (van der Woude et al, 2019), we showed that the transcript levels of endogenous PIF4 were significantly increased in the PIF4‐overexpression transgenic plants compared with the wild type (Fig 7D). Additionally, a recent study reported that BZR1 directly activates the transcriptional expression of PIF4 in high temperature conditions (Ibanez et al, 2018). Therefore, we investigated whether BIC1 cooperates with BZR1/PIF4 to activate the transcriptional expression of PIF4. In the DLR assays, co‐expression of PIF4 or BZR1 with proPIF4:LUC in Arabidopsis protoplasts led a significantly increased LUC activity, confirming that PIF4 or BZR1 can activate the transcription of PIF4 (Fig 7E). As expected, LUC activity was additively enhanced when BIC1 was co‐expressed with PIF4 or BZR1 (Fig 7E). Taken together, these data demonstrate that BIC1 cooperates with BZR1/PIF4 to activate the transcriptional expression of PIF4, revealing a positive feedback loop for transcriptional activation.

The interaction between BIC1 and BZR1 is evolutionally conserved in bread wheat

Bread wheat (Triticum aestivum; 2n = 42; AABBDD) is a major staple crop worldwide. Global demand for bread wheat is increasing with world population growth. Wheat plant architecture improvement is essential to guarantee global food security. Considering that BIC1 interacts with BZR1 to regulate plant growth in the model plant Arabidopsis, we wondered whether BIC1 plays a conserved role in the monocot plants such as bread wheat. To test this idea, three highly homologous genes of bread wheat TaBIC1 were identified based on the Arabidopsis BIC1 gene analysis (Appendix Fig S15). The three TaBIC1 homologous genes were located on chromosomes 2A, 2B, and 2D, respectively. Sequence alignment results showed that bread wheat TaBIC1 shares the common CID domain of Arabidopsis BIC1 (Appendix Fig S16). Subcellular localization assays showed that TaBIC1 was mainly localized in the nucleus (Appendix Fig S17). To investigate whether TaBIC1 interacts with TaBZR1 in bread wheat, a close related BZR1 homolog was identified from bread wheat (Appendix Figs S18 and 19). We performed LCI assays and the results showed that strong interaction signals were observed in the samples co‐expressing nLUC‐TaBIC1 and cLUC‐TaBZR1 (Fig 7F), indicating that TaBIC1 interacts with TaBZR1 in plant cells. In vitro pull‐down assays showed that MBP‐TaBZR1 but not MBP was able to pull down TaBIC1, indicating that TaBIC1 directly interacts with TaBZR1 in vitro (Fig 7G). BiFC assays showed that a strong YFP fluorescence signal was observed in the nucleus when nYFP‐TaBIC1 was co‐transformed with cYFP‐TaBZR1, whereas no signal was observed in the empty vector control (Fig 7H). Taken together, these results indicate that the interaction between BIC1 and BZR1 is conserved in bread wheat.

To determine the biological role of TaBIC1 in bread wheat, we generated the TaBIC1‐overexpression transgenic wheat plants (TaBIC1‐OE) in the wheat cultivar KN199 background which harbors a “Green revolution” gene Rht‐B1b (Peng et al, 1999; Dong et al, 2019). Phenotypic analyses showed that TaBIC1‐OEs exhibited appropriately increased plant height compared with the wheat cultivar KN199 at the mature stage (Appendix Fig S20A and B). RT–qPCR and Western blot analyses confirmed the expression of TaBIC1 transgene (Appendix Fig S20C and D). In Arabidopsis, previous studies have shown that enhanced BR signaling downregulates the expression of BR biosynthetic genes through a negative feedback loop (Wang et al, 2002; He et al, 2005). To test whether overexpression of TaBIC1 affects the BR signaling in bread wheat, we performed RT–qPCR to analyze the expression levels of BR biosynthetic gene TaCPD in the TaBIC1‐OE transgenic wheat plants. The results showed that the expression levels of TaCPD were reduced in the TaBIC1‐OE transgenic wheat plants compared to KN199 (Appendix Fig S20E), suggesting that TaBIC1 positively regulates BR signaling in bread wheat.

Discussion

BZR1 is an atypical basic helix‐loop‐helix (bHLH) transcription factor that binds to thousands of BR‐regulated target genes which contain E‐box (CANNTG) and/or BRRE (BR response element, CGTGT/CG) motifs (He et al, 2005; Vert & Chory, 2006; Sun et al, 2010; Moon et al, 2020). BZR1 was initially identified as a transcriptional repressor that binds to the promoters of BR biosynthetic genes (He et al, 2005). However, a transcription factor usually acts as a transcriptional activator on some target genes promoter and a transcriptional repressor for other target genes. A previous report showed that BZR1 represses target genes by recruiting the Groucho/TUP1‐like transcriptional corepressor TOPLESS (TPL) (Oh et al, 2014b). However, the underlying mechanisms of how BZR1 activates transcription of target genes remain largely unclear.

It has been reported that BIC1 can inhibit the function of CRY2 by blocking blue light‐dependent cryptochrome dimerization and phosphorylation (Wang et al, 2016), but the underlying mechanism of how BIC1 inhibits the phosphorylation of CYR2 is still elusive. In addition, photoregulatory protein kinases (PPKs) interact with CRY2 to catalyze the blue light‐dependent phosphorylation (Liu et al, 2017b). In this scenario, whether BIC1 affects CRY2 phosphorylation through interfering with the interaction between CYR2 and PPKs remains to be elucidated. However, we here provide several lines of evidence showing that BIC1 acts as a transcriptional coactivator for BZR1 to facilitate BR‐induced gene expression and hypocotyl elongation. First, we demonstrated that BIC1 interacts with BZR1 in vitro and in vivo (Fig 1). Second, BIC1 positively regulates BR responses in hypocotyl elongation and BR‐responsive gene expression (Fig 2). Third, genetic evidence showed that BIC1 and BZR1 synergistically promote BR responses (Fig 3). Fourth, we found that BIC1 has transcriptional activation activity to cooperate with BZR1 to activate transcription of target genes (Fig 4B–G). Further, we demonstrate that the transcriptional activation activity of BIC1 is partially required for its function in promoting hypocotyl elongation and target gene expression (Fig 4H and I, Appendix Fig S9C–E). Together, our results suggest that BIC1 acts as a transcriptional coactivator of BZR1 to activate the transcription of cell elongation‐related genes. In contrast, previous studies have shown that BZR1 acts as a transcriptional repressor to repress the transcription of BR biosynthetic genes through recruiting the transcriptional corepressor TPL (He et al, 2005; Oh et al, 2014b). Based on our results, we speculate that the interaction of BIC1 with BZR1 may be sufficient to convert BZR1 from a transcriptional repressor to a transcriptional activator at least partially through the transcriptional activation activity of BIC1. In line with the previous report showing that BICs positively regulate hypocotyl elongation under blue light but not in dark, red light, and far red light (Wang et al, 2016), we here found that the positive regulation of BICs in BR‐induced hypocotyl elongation occurred specifically under blue light, but not in dark and red light (Appendix Fig S4A–F). These observations suggest that BICs may serve as an integration node of blue light and BR signaling to coordinate plant growth.

Previous studies have shown that BZR1 and PIF4 cooperate to activate the expression of cell elongation‐related genes (Bai et al, 2012; Oh et al, 2012). In this study, we showed that BIC1 also interacts with PIF4 to synergistically and interdependently promote hypocotyl elongation and activate target genes, demonstrating that BIC1 acts as a transcriptional coactivator for the transcription factor PIF4 (Fig 5). Consistent with the fact that BIC1 lacks DNA‐binding domain, the physical association of BIC1 with its target genes is dependent on the transcription factors PIFs (Fig 6C). Interestingly, we found that the enrichment of BZR1 and PIF4 to the promoters of target genes requires BICs (Fig 6D and E). Therefore, we propose that BICs might facilitate the accessibility of BZR1/PIF4 to the chromatin of their target genes possibly through recruiting some epigenetic factors for chromatin modification or remodeling.

It is well known that PIF4 is a positive regulator of cell elongation and its activity is regulated by light, temperature, brassinosteroid, and other factors (Choi & Oh, 2016; Martinez et al, 2018). Recent studies showed that BZR1 binds to the promoter of PIF4 to activate its expression at elevated temperature (Ibanez et al, 2018), and PIF4 is also able to bind to its own promoter (van der Woude et al, 2019). Together with our results showing that the transcript levels of endogenous PIF4 were significantly elevated in the PIF4‐overexpression plants (Fig 7D), demonstrating that PIF4 directly activates the transcription of PIF4 itself. Coincidently, our transcriptomic data showed that the transcript levels of PIF4 were also upregulated in the BIC1‐overexpression seedlings (Fig 2D). We further demonstrate that BIC1 associates with the PIF4 promoter and directly activates the transcription of PIF4 (Fig 7A–D). Notably, transient expression assays showed that co‐overexpression of BIC1 and BZR1/PIF4 synergistically enhanced the promoter activity of PIF4 (Fig 7E). Therefore, we conclude that PIF4 is also a common target gene of the transcriptional activation module BIC1‐BZR1‐PIF4, constituting a positive feedback signaling loop for efficient transcriptional activation. As previously reported, the physical association of BZR1/BES1 and other transcription factors such as PIF4 is important for the activation of BR‐induced gene expression (Bai et al, 2012; Oh et al, 2012; Martinez et al, 2018). Therefore, we propose that this positive feedback signaling loop for the transcriptional activation of PIF4 might enhance the formation of the PIF4‐BZR1‐BIC1 complex to activate cell elongation‐related genes.

BICs emerge more recently in land plants and share a highly conserved CID domain in monocot and dicot plants (Wang et al, 2017). To investigate whether BIC1 plays a conserved role in monocot and dicot plants, we determined that the interaction between TaBIC1 and TaBZR1 also occurs in bread wheat (Fig 7F–H). The TaBIC1‐overexpression transgenic wheat plants exhibited appropriately increased plant height compared with the wild type (Appendix Fig S20A and B). The expression levels of BR signaling‐repressive gene TaCPD were reduced in the TaBIC1‐OE transgenic wheat plants (Appendix Fig S20E), suggesting that TaBIC1 also positively regulates BR signaling in bread wheat. Therefore, it will be of interest to explore the utilization of BIC1 homologs in improving crop plant architecture using natural variation selection or CRISPR/Cas9 technology.

In conclusion, we demonstrate that BIC1 positively regulates BR signaling and acts as a transcriptional coactivator for the transcription factors BZR1 and PIF4 to synergistically and interdependently activate the transcription of common target genes including PIF4 and other cell elongation‐related genes, consequently promoting cell elongation (Fig 7I). Our data also provide new clues for further dissection of the molecular mechanisms underlying the crosstalk of BR and light signaling.

Materials and Methods

Plant materials and growth conditions

All the Arabidopsis plants used in this study are in Col‐0 ecotype background. The following transgenic and mutant lines used in this study have been described previously: bic1bic2 (Wang et al, 2016), 35S:BZR1‐MYC (Wang et al, 2012a), bzr1‐1D (Wang et al, 2002), 35S:PIF4 (de Lucas et al, 2008), 35S:PIF4‐HA (de Lucas et al, 2008), pifQ (Shin et al, 2009), and pPIF4:GUS (Sun et al, 2013). For constitutive overexpression, full‐length BIC1 coding sequence was cloned into pEarly‐101 or pCambia1300 to generate 35S:BIC1‐YFP and 35S:BIC1‐Flag. The fragment containing the BIC1 promoter (1,832 bp) and the coding region was amplified from Col‐0 genomic DNA and ligated into the pCAMBIA1300 vector to generate the pBIC1:BIC1 transgenic plants. For the generation of β‐estradiol‐inducible pERGW‐BIC1, CDS of BIC1 was cloned into pERGW vector. To generate BIC1-SRDX‐Flag, the C‐terminal of BIC1 gene was fused with the transcriptional repressor domain SRDX (LDLDLELRLGFA) and then cloned into pCambia1300 vector. The double transgenic plant 35S:BZR1‐MYC/35S:BIC1‐YFP was obtained by genetic crossing between 35S:BIC1‐YFP and 35S:BZR1‐MYC. The double transgenic plant pBIC1‐BIC1/35S:BZR1‐MYC was obtained by genetic crossing between pBIC1:BIC1 and 35S:BZR1‐MYC. 35S:BZR1‐MYC/bic1bic2 was obtained by genetic crossing 35S:BZR1‐MYC and bic1bic2. The double transgenic plant 35S:PIF4‐MYC/35S:BIC1‐YFP was obtained by genetic crossing 35S:BIC1‐YFP and 35S:PIF4‐MYC. The double transgenic plant pBIC1‐BIC1/35S:PIF4‐MYC was obtained by genetic crossing between pBIC1:BIC1 and 35S:PIF4‐MYC. 35S:PIF4‐MYC/bic1bic2 was obtained by genetic crossing 35S:PIF4‐MYC and bic1bic2. pPIF4:GUS/35S:BIC1‐YFP was obtained by genetic crossing pPIF4:GUS and 35S:BIC1‐YFP. 35S:BIC1‐Flag/pifQ #7, #8 were obtained by transforming the 35S:BIC1‐Flag transgene into the pifQ mutant background. Arabidopsis and Nicotiana benthamiana plants were grown in a greenhouse set at 22°C and a long‐day conditions (16‐h light/8‐h dark) for general growth and seed harvesting. The bread wheat cultivar Kenong 199 (KN199) was used for gene cloning and genetic transformation. The TaBIC1‐OE transgenic wheat lines used in this study are in KN199 background. For field experiments, wheat plants were grown in the experimental station of the Institute of Crop Sciences, CAAS, Beijing, under natural growth conditions.

DNA constructs

For Gateway cloning, all the gene sequences were cloned into the pQBV3 (Gateway) and subsequently introduced into certain destination vectors following the Gateway technology (Invitrogen). For ligase‐dependent cloning, the endonuclease digested vectors and PCR fragments were separately purified by PCR cleanup kit (Axygen, AP‐PCR‐250), and ligated at 16 h with T4 DNA ligase (New England Biolabs, M2020). For ligase‐independent ligation, the ligation free cloning master mix (abm) was used following the application handbook. Oligo primers used for cloning are listed in Appendix Table S1.

Hypocotyl measurement

Seeds were surface‐sterilized for 7 min with 70% (v/v) ethanol and washed for 3 min with 100% (v/v) ethanol, and then sown on half‐strength Murashige and Skoog (MS) medium. After 2‐day vernalization, the plates were placed under long‐day, 22°C conditions for 5–6 days. Seedlings were photocopied, and hypocotyl lengths were measured by using ImageJ software (http://rsb.info.nih.gov/ij).

RNA extraction and real‐time PCR

Total RNAs were extracted using a Plant Total RNA extraction kit (Zoman) and reverse‐transcribed to cDNA using an abm reverse transcriptase kit, according to the manufacturer's instructions. Reverse transcription–quantitative PCR (RT–qPCR) was performed by using SYBR Premix Ex Taq (Perfect Real Time; Takara) on LightCycler 96 (Roche). The gene expression results were normalized by PP2A. For the wheat gene expression, the expression levels of target genes were normalized to TaGAPDH. All the experiments were performed independently three times. Primers are listed in Appendix Table S1.

RNA‐sequencing and data analysis

Col‐0, 35S:BIC1‐YFP seedlings grown under long‐day conditions chamber for 10 days and were collected. Three replicates were prepared for each sample. RNA was extracted by TRIzol reagent (Invitrogen). Libraries were constructed by and further sequenced on Illumina HiSeq. 2000 platform (Illumina Inc., USA) with three independent biological replications for each sample. After screening and trimming, clean reads were mapped to the Arabidopsis thaliana genome (TAIR10, www.arabidopsis.org) using the TopHat2 software. Cufflinks methods were used for determination of expression values. Genes with estimated absolute fold changes ≥ 1.5 were identified as reliable differentially expressed genes (DEGs). Morpheus (https://software.broadinstitute.org/morpheus) was used to construct heat maps. PlantGSEA (http://structuralbiology.cau.edu.cn/PlantGSEA/index.php) was used for GO term enrichment analysis. http://bioinformatics.psb.ugent.be/webtools/Venn/ online tool was used to perform the comparisons of DEGs.

Immunoblotting

Protein extraction and immunoblotting were carried out as described previously (He et al, 2019). Seedlings were grown under long‐day conditions for 5 days and then were treated for various time or not. The protein abundance of BIC1‐YFP or BIC1‐Flag was detected with an anti‐GFP (Roche, catalog number 11814460001) or an anti‐Flag antibody (MBL, catalog number M185‐3), respectively. The protein levels of BZR1‐MYC or PIF4‐MYC were detected with an anti‐MYC antibody (Roche, catalog number 1167149001). Actin (CWbiotech, catalog number CW0264) was used an internal control.

Transactivation activity assays in yeast

The transactivation activity assay was carried out as previously described (Liu et al, 2017a). BIC1 and its truncated versions (including the NT and CT) were fused with the GAL4‐BD vector pGBKT7 and transformed into the yeast strain Gold according to the instructions for the Frozen‐EZ Yeast Transformation II Kit (ZYMO RESEARCH). The transformants were first selected on synthetic dextrose growth medium lacking Trp (SD‐W). Then, the yeast strains were dropped on synthetic dextrose selection medium lacking Trp and His (SD‐W/H) for transcriptional activation activity evaluation according to their growth status.

Chromatin immunoprecipitation (ChIP) assays

ChIP experiments were performed as described previously (Liu et al, 2017a). The 6‐day‐old Col‐0, 35S:BIC1‐Flag, 35S:BIC1‐Flag/pifQ, 35S:PIF4‐MYC and 35S:PIF4‐MYC/bic1bic2 seedlings and the 10‐day‐old 35S:BZR1‐MYC and 35S:BZR1‐MYC/bic1bic2 seedlings with 1 μM eBL treatment for 3 h were used for the ChIP assays. About 2 g of each sample was harvested and cross‐linked in 1% formaldehyde for 15 min, followed by 5‐min neutralization with 0.125 M glycine. After washing for 5 times with distilled water, the seedlings were grounded to powder in liquid nitrogen and the chromatin complexes were isolated and sonicated and then the chromatin complex was immunoprecipitated by anti‐MYC antibody (Abcam ChIP grade, catalog number ab9132) or anti‐Flag antibody (MBL, catalog number M185‐3). The precipitated DNA was recovered and analyzed by real‐time qPCR using the respective primer pairs listed in Appendix Table S1.

BiFC assays

The coding sequences of BIC1 and BZR1 were cloned into the pEarleyGate 201‐YN (nYFP‐BIC1) or pEarleyGate 202‐YC vectors (cYFP‐BZR1) using Gateway system (Ju et al, 2019). The prepared vectors were introduced into Agrobacterium strain GV3101 and then co‐expressed in Nicotiana benthamiana leaves. After 48 h after infiltration, the fluorescence signal of yellow fluorescent protein (YFP) was observed with confocal microscopy (Carl Zeiss, LSM880). Images were captured at 514 nm laser excitation and 519‐620 nm emission for YFP. Three biological replications were performed.

Pull‐down assays

The coding sequence of BIC1, BZR1, or PIF4 was cloned into pGEX‐4T-1, pMAL‐C2X, or pMAL‐C5X vectors. GST‐BIC1, MBP‐PIF4, and MBP‐BZR1 proteins were expressed in E. Coli (BL21) and were purified using GST Bind Resin (Millipore) or amylose resin (NEB). For pull‐down assays, amylose resin was used to pull down protein complexes in binding buffer (25 mM Tris–HCl [pH 7.5], 100 mM NaCl, 1 mM DTT, Roche protease inhibitor cocktail) at 4°C for 2 h. Then, the beads were collected and washed five times with washing buffer (25 mM Tris–HCl [pH 7.5], 150 mM NaCl, 0.1% Triton X‐100, 1 mM DTT). Subsequently, samples were boiled in SDS–PAGE sample buffer, and the elutants were analyzed by immunoblot probed with anti‐MBP (CWbiotech, catalog number CW0288) or anti‐GST (CWbiotech, catalog number CW0144) antibody.

Co‐immunoprecipitation assays

The transgenic plants 35S:YFP, 35S:BIC1‐YFP, 35S:BZR1‐MYC, and 35S:BZR1‐MYC/35S:BIC1‐YFP were used for detecting the interaction between BIC1 and BZR1, and for the interaction between BIC1 and PIF4, the transgenic plants 35S:BIC1‐YFP, 35S:PIF4‐MYC and 35S:PIF4‐MYC/35S:BIC1‐YFP were used. Seedlings were grown in a long‐day, 22°C growth chamber for 7 days. Samples were harvested and grounded to fine powder in liquid nitrogen, and homogenized in IP buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA [pH 8.0], 1% Triton X‐100, 0.6 mM PMSF, 20 μM MG132, Roche protease inhibitor cocktail). The extracts were centrifuged at 14,000 g for 20 min. The supernatant was mixed with 20 μl Dynabeads protein G Beads (Invitrogen catalog number 10003) to reduce nonspecific immunoglobulin binding for 1 h. After a brief spin, the supernatant was incubated with 30 μl anti‐MYC magnetic beads (MBL catalog number M047‐10) overnight at 4°C and then washed 5 times with IP buffer. Samples were boiled in SDS–PAGE sample buffer, and the elutants were analyzed by immunoblot probed with anti‐GFP or anti‐MYC antibody.

LCI assays

The LCI assays for the protein interaction detection were performed in N. benthamiana leaves as described previously (Sun et al, 2013; Dong et al, 2019). Briefly, the full‐length or truncated forms of the genes were cloned into vectors pCAMBIA1300‐nLUC or pCAMBIA1300‐cLUC and transformed to Agrobacterium strain GV3101 and then co‐infiltrated into Nicotiana benthamiana leaves, and the LUC activities were analyzed after 48‐h infiltration using NightSHADE LB 985 (Berthold).

Transient expression assays in Arabidopsis protoplasts

The transient transcriptional activity analysis assay was performed in Arabidopsis protoplast as described previously (Liu et al, 2018). The coding sequence of BIC1 was cloned into the effector GAL4‐DB vector. Two types of reporters were one contains LUC gene fused with 5xGAL4 binding site and the other is a plasmid expressing the REN gene as the internal control. The effector plasmids (GAL4‐DB-BIC1 or positive control GAL4‐DB-VP16) were co‐transformed with two reporters, and the activities of LUC and REN were separately determined 18 h post‐transformation using Dual‐Luciferase^® Reporter Assay System (Promega, E1910).

For the transient transcriptional activity assays on specific promoter, the PRE5 and PIF4 promoter sequences were amplified from Col‐0 genome DNA and cloned into the pGreenII 0800‐LUC vector as a reporter and the REN gene under the control of the cauliflower 35S promoter in the pGreenII 0800‐LUC vector was used as the internal control. The coding sequences of YFP, BIC1, BZR1, and PIF4 were cloned into p2GW7 vector under the control of the 35S promoter and were used as effectors. The reporters and effectors were transfected into Arabidopsis protoplasts in different combinations and incubated in dark for 18 h.

Subcellular localization analysis

The coding sequence of TaBIC1 was cloned into the pEarly‐101 vector and then was transformed into Agrobacteriu strain GV3101. The Agrobacterium was infiltrated into Nicotiana benthamiana leaves, and the fluorescence signal of yellow fluorescent protein (YFP) was observed at 48 h post‐infiltration.

Accession numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or in the Triticeae Multi‐omics Center (http://202.194.139.32/) with the following accession numbers: AT1G75080 (BZR1), AT3G52740 (BIC1), AT2G46830 (CCA1), AT5G61380 (TOC1), AT3G46640 (LUX), AT2G25930 (ELF3), AT5G39860 (PRE1), AT3G28857 (PRE5), AT4G38850 (SAUR‐AC), AT1G29490 (SAUR68), AT2G06850 (XTH4), AT1G20190 (EXP11), AT1G04240 (IAA3), AT1G65480 (FT), AT1G01060 (LHY), AT3G15540 (IAA19), AT4G32280 (IAA29), AT4G28720 (YUC8), AT2G43010 (PIF4), AT1G13320 (PP2A), TraesCS2A02G294600 (TaBIC1‐A), TraesCS2B02G311000 (TaBIC1‐B), TraesCS2D02G292300 (TaBIC1‐D), TraesCS2A02G187800 (TaBZR1), and TraesCS5A02G131400.1 (TaCPD).

Author contributions

JS and ZY designed research; ZY, BY, HD, and GH performed research; and ZY, YZ, and JS analyzed the data and wrote the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Supporting information

Appendix

Source Data for Appendix

Review Process File

Source Data for Figure 1

Source Data for Figure 4

Source Data for Figure 5

Source Data for Figure 7

The EMBO Journal (2021) 40: e104615

Data availability

The RNA‐sequencing data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO): (accession GSE150362, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE150362).