The G-protein γ subunit DEP1 facilitates brassinosteroid signaling in rice via a MYB–bHLH–ARF module

Abstract

G-protein signaling and brassinosteroid (BR) phytohormones play important roles in regulating rice (Oryza sativa) yield-related plant architecture, such as leaf inclination and grain size. However, the relationship between G‐proteins and BR signaling has not been fully elucidated in rice. The present study indicates that the G‐protein Gγ subunit DENSE AND ERECT PANICLE 1 (DEP1) positively regulates BR signaling in rice and that BRs promote DEP1 nuclear entry through GRAIN NUMBER ASSOCIATED (GNA). Additionally, DEP1 interacts with and acts upstream of OsMYB86, an R2R3-MYB family transcription factor that positively regulates BR signaling by directly binding to the promoter of its downstream gene BRASSINOSTEROID UPREGULATED 1 (BU1), activating its expression in rice. In the nucleus, DEP1 interacts with OsMYB86 and GNA, significantly enhancing OsMYB86-mediated activation of BU1 expression. Furthermore, BU1 interacts with another HLH protein, INCREASED LEAF INCLINATION1 (ILI1), and a bHLH protein, ILI1 BINDING bHLH (IBH1). Interaction between ILI1 and BU1 facilitates translocation of BU1 from the cytoplasm to the nucleus, where they impede IBH1 binding to the promoter of the AUXIN RESPONSE FACTOR 11 (OsARF11) gene, which is involved in crosstalk between BR and auxin, thus effectively relieving the IBH1-repressed transcription of OsARF11. These findings reveal a DEP1-mediated signaling pathway that links G-proteins to the traditional BR signaling pathway, ensuring the efficient activation of BR responses in rice.

A complex pathway involving the G-protein γ subunit DENSE AND ERECT PANICLE 1 transduces brassinosteroid (BR) signals, leading to the efficient activation of BR responses.

Introduction

Leaf inclination, the inclination between the leaf blade and the culm, is an important character affecting rice plant architecture (Zhou et al. 2017). In rice plants erect leaves can enhance photosynthetic efficiency, nitrogen storage, and planting density; thus, they have great potential for improving rice productivity (Sakamoto et al. 2006). Leaf inclination is mainly controlled by lamina joint development (Zhou et al. 2017), which is influenced by several factors including plant hormones (Sun et al. 2015), soil phosphorus content (Ruan et al. 2018; Guo et al. 2022), mechanical tissues (Ning et al. 2011; Huang et al. 2021), and gravitropism (Morita and Tasaka 2004).

Stimulating lamina joint development is a typical effect of brassinosteroids (BRs), a class of steroid hormones found in plants (Tanabe et al. 2005; Sakamoto et al. 2006; Sun et al. 2015). There is growing evidence that many other phytohormones, including auxin (Qiao et al. 2022), gibberellin (Shimada et al. 2006), and abscisic acid (Li et al. 2019, 2021) act synergistically or antagonistically with BRs to influence leaf inclination in rice. BRs are also involved in various plant growth and development processes such as cell expansion and division, floral organogenesis, leaf growth, grain development, and resistance to biotic and abiotic stresses (Bishop and Koncz 2002; Nakashita et al. 2003; Tong et al. 2012; Tong and Chu 2018; Nolan et al. 2020). Patterns of BR signaling and biosynthesis pathways in plants have been gradually revealed over the last few decades (Zhao and Li 2012; Kim and Russinova 2020).

Several crucial enzymes that biosynthesize BRs can affect BR levels in plants, leading to changes in leaf inclination. The BRASSINOSTEROID-DEFICIENT DWARF2 (BRD2) gene encodes a protein with FAD-linked oxidoreductase activity, and a mutant form resulted in a typical BR-deficient phenotype with erect leaves (Hong et al. 2005). Ebisu Dwarf (D2), Brassinosteroid-deficient dwarf1 (BRD1), Dwarf11 (D11), and Dwarf4 encode members of the cytochrome P450 family, and loss of their functions inhibits BR biosynthesis, resulting in smaller leaf inclination (Mori et al. 2002; Hong et al. 2003; Tanabe et al. 2005; Sakamoto et al. 2006).

In Arabidopsis thaliana BRs interact with the receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1) (Hothorn et al. 2011) and its coreceptor BRI1-ASSOCIATED RECEPTOR KINASE 1. The binding of BRs to BRI1 induces BRI1 KINASE INHIBITOR 1 disassociation and transphosphorylation between BRI1 and its coreceptor BAKs, leading to phosphorylated BSK release from BRI1 (Wang and Chory 2006). Phosphorylated BSK proteins catalyze the phosphorylation and activation of BRI1-SUPPRESSOR 1 (BSU1) (Tang et al. 2008; Kim et al. 2011). Activated BSU1 dephosphorylates and inactivates Brassinosteroid-Insensitive 2 (BIN2), which functions as a repressor of BR signaling and inhibits BR responses by phosphorylating Brassinazole-Resistant 1/2 (BZR1/2), resulting in their transport out of the cytoplasm with the help of 14-3-3 protein (Li and Nam 2002; Bai et al. 2007; Gampala et al. 2007; Clouse 2011). Loss of BIN2 activity leads to dephosphorylation of BZR1/2. Lastly, dephosphorylated BZR1/2 accumulates in the nucleus, and regulates the expression of BR-responsive genes (He et al. 2005; Sun et al. 2010).

In rice, OsBRI1 and OsBAK1 function as BR receptors and coreceptors, and loss-of-function OsBRI1 and OsBAK1 mutants exhibit an erect leaf phenotype (Yamamuro et al. 2000; Park et al. 2011). Knockdown of GLYCOGEN SYNTHASE KINASE3/SHAGGY-like kinase (GSK2), a rice homolog of BIN2, enhances OsBZR1 transcriptional activity and leaf inclination (Qiao et al. 2017). Several transcription factors including GROWTH-REGULATING FACTOR4 (GRF4) (Che et al. 2015; Duan et al. 2015), SMALL ORGAN SIZE1 (SMOS1)/REDUCED LEAF ANGLE1 (RLA1) (Qiao et al. 2017), DWARF AND LOW-TILLERING (DLT) (Tong et al. 2012) and OVATE FAMILY PROTEIN1 (Xiao et al. 2017) function downstream of GSK2 and positively regulate leaf inclination in rice. TAIHU DWARF1 encodes a U-box family E3 ubiquitin ligase that promotes BR signaling by interacting with the G-protein alpha subunit RGA1 and GSK2 (Hu et al. 2013; Liu et al. 2023). GNA/DLT2/OsGRAS19 encodes a GRAS-type transcription factor and participates in BR signaling by interacting with DLT and BZR1 (Chen et al. 2013; Zou et al. 2023; Zhang et al. 2024). Downstream of OsBZR1, the basic helix-loop-helix (bHLH) transcription factors ILI1 and IBH1 act antagonistically to regulate rice leaf inclination (Zhang et al. 2009a). LIC and BZR1 antagonistically regulate the expression of ILI1 and IBH1 (Zhang et al. 2012). BU1 encodes an HLH transcription factor lacking DNA-binding ability, and has been proposed to positively regulate BR signaling (Tanaka et al. 2009). There are many MYB genes in rice, and they also participate in BR signaling (Yanhui et al. 2006; Feller et al. 2011). For example, REGULATOR OF LEAF INCLINATION 1 and OsGAMYBL2 regulate leaf inclination in rice by regulating the expression of BU1 and BU1-LIKE1 COMPLEX1 (Gao et al. 2018; Ruan et al. 2018). Although BU1, ILI1, IBH1, and some MYB family genes reportedly regulate leaf inclination and BR signaling in rice, their specific functions have not been fully elucidated. Further studies are needed to clarify their specific roles.

G-proteins, composed of Gα, Gβ, and Gγ subunits, serve as signal transduction hubs in both plant and animal cells (Urano et al. 2013; Urano and Jones 2014). The G-protein alpha subunit RGA1 has been implicated in both BR and GA signaling pathways in rice (Ueguchi-Tanaka et al. 2000; Wang et al. 2006). The Gβ subunit RGB1 promotes ABA biosynthesis, and the Gγ subunit DEP1 represses ABA responses (Zhang et al. 2015a). DEP1 interacts with Gα subunit RGA1 and Gβ subunit RGB1, and reduced RGA1 activity or increased RGB1 activity leads to nitrogen response inhibition (Sun et al. 2014, 2018). Rice varieties with dominant dep1 mutation exhibit typical erect leaves (Huang et al. 2009; Sun et al. 2018). DEP1‐overexpressing plants also exhibit a notably increased leaf inclination phenotype (Sun et al. 2018). DEP1 cooperatively activates the expression of REGULATOR OF LEAF ANGLE (OsRELA)/DENSE AND ERECT PANICLE 2 (DEP2) by interacting with BR signaling pathway transcription factor SMOS1/RLA1/GRAIN ROUND 5 (Qiao et al. 2017; Wang et al. 2024). OsRELA/DEP2 interacts with the BR signaling negative regulator LEAF AND TILLER ANGLE INCREASED CONTROLLER (OsLIC), inhibiting its transcriptional activity, thus promoting leaf inclination in rice (Zhu et al. 2021). These findings suggest that DEP1 likely plays a role in the BR signaling pathway, but the detailed pathway by which DEP1 regulates BR signaling remains unclear.

Here, we found that through GNA, BRs promote DEP1's entry into the nucleus, where DEP1 interacts with OsMYB86, activating OsMYB86-mediated transcription of BU1. Further, ILI1 interacts with BU1 and promotes the translocation of BU1 from the cytoplasm to the nucleus, where together they alleviate IBH1-repressed transcription of OsARF11, thus promoting BR signaling. Thus, these findings reveal a DEP1-GNA-OsMYB86- BU1/ILI1/IBH1-OsARF11 signaling pathway that intricately connects the G-protein subunit DEP1 with the BR signaling pathway in rice.

Results

DEP1 is a positive regulator of BR signaling

To investigate the role of DEP1 in BR signaling, we created DEP1 knockout (dep1-1/2) and DEP1 overexpressing (DEP1-OE) lines, in the rice variety Kitaake (Supplementary Fig. S1). The dep1-1/2 plants showed erect flag leaves with reduced lamina inclination, while the DEP1-OE plants displayed flag leaves with increased lamina inclination compared with wild type (WT) plants (Fig. 1, A and B). Consistent with previous reports (Sun et al. 2018; Wang et al. 2024), dep1-1 and dep1-2 plants had smaller grain length, whereas DEP1-OE plants had larger grain length (Fig. 1, C and D). In lamina inclination assays, dep1-1 and dep1-2 plants exhibited reduced sensitivity to 2,4-epibrassinolide (2,4-epiBL) treatment compared with WT, whereas DEP1-OE plants exhibited enhanced sensitivity (Fig. 1, E and F). In coleoptile elongation assays dep1-1 was also less sensitive to 2,4-epiBL treatment than WT (Supplementary Fig. S2). In RNA-Seq experiments, Gene Ontology (GO) enrichment analysis revealed that the DEGs between dep1-1 and WT were enriched for annotated biological functions associated with hormone response, hormone-mediated signaling pathways, and hormone metabolic processes (Supplementary Fig. S3). A heatmap showed that, compared with WT, the expression levels of several genes involved in BR signaling and biosynthesis were altered in dep1-1 and DEP1-OE transgenic plants (Supplementary Fig. S4). Expression of OsHLH92 (Teng et al. 2023), OsOFP22 (Chen et al. 2021), and ILI1 (Zhang et al. 2009a), 3 BR positive response genes, was significantly increased by 2,4-epiBL treatment in WT, but it was suppressed in dep1-1 mutants (Supplementary Fig. S5, A to C). Expression levels of IBH1 (Zhang et al. 2009a), OsGRF4 (Duan et al. 2015), and DLT (Tong et al. 2009)—3 BR negative response genes—were downregulated by 2,4-epiBL treatment in WT plants, but these responses were also disrupted in dep1-1 mutants (Supplementary Fig. S5, D to F). Expression levels of D2, D11, and BRD2 were downregulated by 2,4-epiBL treatment in WT, but they were upregulated in dep1-1 mutants (Supplementary Fig. S5, G to J). This is consistent with previous reports that expression of some BR synthesis genes was downregulated by BL due to BR feedback inhibition (Tong et al. 2009; Qiao et al. 2017), and suggests that loss of DEP1 function likely interferes with the normal BR feedback inhibition pathway. Substantial upregulation of DEP1 expression was observed after 1 h of treatment with 1 μM 2,4-epiBL (Fig. 1G), indicating that DEP1 plays a positive role in regulating flag leaf inclination in rice by responding to BRs.

Figure 1.

DEP1 is a positive regulator of BR signaling. A) Plant phenotypes of Kitaake, dep1-1, dep1-2 and DEP1-OE at mature stage. Bar = 15 cm. B) Phenotypes and measurements of the flag leaf inclination of Kitaake, dep1-1, dep1-2, and DEP1-OE at mature stage. Asterisks indicate significant difference compared with Kitaake. Data = means ± SD (n = 12, **P < 0.01, Student's t-test). Bar = 4 cm (applies to all images in Panel B). C and D) Phenotypes and measurements of the grain length of Kitaake, dep1-1, dep1-2, and DEP1-OE. Asterisks indicate significant difference compared with Kitaake. Data = means ± SD (n = 20, **P < 0.01, Student's t-test). Bar = 10 mm. E) Lamina bending analysis of Kitaake, dep1-1, dep1-2, and DEP1-OE at the seedling stage in response to BL (brassinolide). Bar = 4 cm (applies to all images in Panel E). F) Quantification of the lamina inclination bending assay in (E) in response to different concentrations of BL. Data = means ± SD. The percentages indicate the promoting effect of BL on lamina inclination. Different letters indicate significant differences as determined by Tukey's multiple comparisons test (P < 0.05, n = 14). G) The expression change pattern of DEP1 in response to 1 μM BL. Data = means ± SD. Ethanol was used as a mock treatment. The different letters above the histogram indicate significant differences (P < 0.05) by Tukey's multiple comparison test. (n = 3).

BRs promote DEP1 nuclear entry through GNA

Many previous studies have identified different subcellular DEP1 localization patterns (Huang et al. 2009; Zhou et al. 2009; Taguchi-Shiobara et al. 2011; Sun et al. 2014, 2018; Liu et al. 2018; Matsuta et al. 2018; Miao Liu et al. 2021; Wang et al. 2024). Some previous studies have shown that DEP1 can be localized in the nucleus (or translocated) into the nucleus to interact with a number of transcription factors (Liu et al. 2018; Miao Liu et al. 2021; Wang et al. 2024). Our recent findings revealed that GNA can effectively facilitate the nucleus entry of DEP1 (Zhang et al. 2024). Additionally, previous research has demonstrated that BRs promote the accumulation of GNA proteins in both Nicotiana benthamiana and rice (Chen et al. 2013; Zou et al. 2023). To explore the detailed mechanism by which BRs regulate the subcellular localization of DEP1, we analyzed its localization in rice protoplasts and N. benthamiana leaves. Three DEP1-GFP localization types were observed in Nipponbare protoplasts, with different percentages; type I, cytoplasmic and membrane localization without nuclear localization signals of DEP1-GFP (41%), type II, cytoplasmic and membrane localization with nuclear membrane outline (43%), and type III, cells with evident nuclear localization (16%) (Fig. 2, A and D). Only 2 localization types, type I (50%) and type II (50%), were observed in protoplasts of the BR synthesis-deficient mutant brd1, and DEP1-GFP did not show clear nuclear localization in brd1 protoplasts (Fig. 2, B and D). However, co-expression of DEP1-GFP with GNA-FLAG in brd1 protoplasts restored the nuclear localization of DEP1-GFP (Fig. 2, C and D). Western blot analysis further validated these observations (Fig. 2E). After transforming the leaf epidermal cells of N. benthamiana with Agrobacterium for 60 to 72 h, DEP1-GFPs are localized in the membrane and cytoplasm, with only approximately 4% cells exhibiting an evident nuclear membrane outline (Supplementary Fig. S6A). Co-expression of DEP1-GFP and GNA-FLAG resulted in nuclear localization of DEP1-GFP in approximately 14% cells (Supplementary Fig. S6, B and E). BL treatment resulted in relatively weak nuclear localization of DEP1-GFP in approximately 20% cells (Supplementary Fig. S6, C and E). Following BL treatment, approximately 35% of leaf epidermal cells co-expressing DEP1-GFP and GNA-FLAG exhibited the most prominent nuclear localization signals of DEP1-GFP. (Supplementary Fig. S6, D and E). Western blot analysis further confirmed that the combination of BL and GNA-FLAG exhibited the highest efficiency in promoting DEP1 nuclear localization in N. benthamiana. (Supplementary Fig. S6F). Based on these findings, we conclude that BRs facilitate DEP1 nuclear entry via GNA.

Figure 2.

BRs promote DEP1 nuclear entry through GNA. A) Three localization patterns of DEP1-GFP in Nipponbare protoplasts. Type I, cytoplasmic and membrane localization without nuclear localization signals of DEP1-GFP. Type II, cytoplasmic and membrane localization with nuclear membrane outline. Type III, cells with evident nuclear localization. B) Two localization patterns of DEP1-GFP in brd1 mutant protoplasts. C) Two localization patterns of DEP1-GFP co-expressed with GNA in brd1 mutant protoplasts. D) The proportion of DEP1-GFP localization in different expression combinations. 100 cells were counted for each expression combination. E) The nuclear and cytoplasmic distribution of DEP1-GFP in different expression combinations. Histone 3 and actin were used as markers for the nucleus and cytoplasm, respectively. C, cytoplasmic fraction; N, nuclear fraction. D53-mCherry, a nuclear marker. Bar = 10 μm.

DEP1 interacts with OsMYB86

To delve deeper into the molecular mechanism of DEP1 involvement in BR signaling, we conducted a yeast 2-hybrid screening and identified a candidate protein, OsMYB86 (Figs. 3A and S7). OsMYB86 is a typical transcription factor of the R2R3-MYB family, which is widely expressed in different tissues, with high expression levels in the panicles of rice (Supplementary Figs. S8 to S10). Interaction between DEP1 and OsMYB86 was further verified by a bimolecular fluorescence complementation (BiFC) assay (Fig. 3B), a luciferase complementation imaging (LCI) assay (Fig. 3C), and a co‐immunoprecipitation (Co-IP) assay in N. benthamiana leaves (Fig. 3D). As well as DEP1, OsMYB86 also interacted with 2 other Gγ subunits, GS3 and GGC2, but did not interact with the Gα subunit (RGA1), the Gβ subunit (RGB1), or GNA (Figs. 3A and S11). Co-expression of DEP1-GFP with OsMYB86-FLAG in N. benthamiana leaves led to nuclear localization of DEP1-GFP in approximately 4% of the cells (Supplementary Fig. S12). In comparison with protoplasts derived from WT Kitaake, the percentage of type III cells expressing DEP1-GFP in the osmyb86 mutant protoplasts diminished from 11% to 4% (Supplementary Fig. S13). These results indicate that OsMYB86 can interact with DEP1, but its capacity to facilitate the nuclear translocation of DEP1 is significantly weaker than that of GNA.

Figure 3.

DEP1 interacts with OsMYB86. A) OsMYB86 interacts with DEP1, GS3, and GGC2, but not with RGA1 or RGB1 in yeast cells. SD-Leu-Trp, selective medium lacking Leu and Trp. SD-Leu-Trp-Ade-His, lacking Trp, Leu, His, and Ade. pGADT7-DLT and pGBKT7-RGA1 were used as the negative controls. B) BiFC assay verifies the interaction between DEP1 and OsMYB86 in the leaf epidermal cells of N. benthamiana. P2YN-RGA1 and P2YC-DLT were used as the negative controls. Arrowheads represent nuclear localization. Bar = 50 μm. C) LCI assay verifies that DEP1 interacts with OsMYB86 in the leaf epidermal cells of N. benthamiana. RGA1-nLUC and DLT-cLUC were used as the negative controls. ILI1-nLUC and IBH1-cLUC were used as the positive controls. Colored scale bar indicates the luminescence intensity in CPS. D) Co-IP analysis of the interaction between DEP1-GFP and OsMYB86-FLAG in the leaf epidermal cells of N. benthamiana. IB, immunoblotting analysis. kDa, kilodaltons.

OsMYB86 positively regulates BR signaling by directly activating BU1 expression

To investigate whether OsMYB86 is related to BR signaling, knockout lines (osmyb86-1,2,3) and overexpressing lines (OsMYB86-OE-1,2,3) of Kitaake were generated (Supplementary Fig. S14). Compared with WT, flag leaf inclination and grain length were reduced in osmyb86-1,2,3 plants, whereas they were increased in OsMYB86-OE plants (Fig. 4, A to D). Osmyb86-1,2,3 plants exhibited no significant changes in grain width or grain thickness. One OsMYB86-OE line exhibited increased grain width, and 2 OsMYB86-OE lines exhibited increased grain thickness (Supplementary Fig. S15). Compared with WT, osmyb86-1 and osmyb86-3 plants were hyposensitive to 2,4-epiBL, whereas OsMYB86-OE-1 and OsMYB86-OE-2 plants were hypersensitive to 2,4-epiBL (Fig. 4, E and F). OsMYB86 expression was induced after 1 h of treatment with 1 μM 2,4-epiBL, and 12 h of treatment with 1 μM 2,4-epiBL (Fig. 4G). These results indicate that OsMYB86 positively regulates BR signaling, controlling flag leaf inclination in rice.

Figure 4.

OsMYB86 positively regulates BR signaling as a BR-responsive factor. A) Plant phenotypes of Kitaake, osmyb86-1, osmyb86-2 and osmyb86-3 at mature stage. Bar = 15 cm. B) Plant phenotypes of Kitaake, OsMYB86-OE-1, OsMYB86-OE-2, and OsMYB86- OE-3 at mature stage. Bar = 15 cm. C) Phenotypes and measurements of the flag leaf inclination of Kitaake, osmyb86-1, osmyb86-2, osmyb86-3, OsMYB86-OE-1, OsMYB86-OE-2, and OsMYB86-OE-3 at mature stage. Asterisks indicate significant difference compared with Kitaake. Data = means ± SD (n = 15, **P < 0.01, *P < 0.05, Student's t-test). Bar = 4 cm (applies to all images in Panel C). D) Phenotypes and measurements of the grain length of Kitaake, osmyb86-1, osmyb86-2, osmyb86-3, OsMYB86-OE-1, OsMYB86-OE-2, and OsMYB86-OE-3. Asterisks indicate significant difference compared with Kitaake. ns indicates no significance compared with Kitaake. Data = means ± SD (n = 10, **P < 0.01, *P < 0.05, Student's t-test). Bar = 10 mm. E) Lamina bending analysis of Kitaake, osmyb86-1, osmyb86-3, OsMYB86-OE-1, and OsMYB86-OE-2 at the seedling stage in response to BL. Bar = 4 cm (applies to all images in Panel E). F) Quantification of the lamina inclination bending assay in (E) in response to different concentrations of BL. Data = means ± SD. The percentages indicate the promoting effect of BL on lamina inclination. Different letters indicate significant differences as determined by Tukey's multiple comparisons test (P < 0.05, n = 15). G) The expression change pattern of OsMYB86 in response to 1 μM BL. Ethanol was used as a mock treatment. Data = means ± SD. The different letters above the histogram indicate significant differences (P < 0.05) by Tukey's multiple comparison test. (n = 3).

To identify OsMYB86's direct downstream targets, chromatin immunoprecipitation-sequencing (ChIP-seq) analysis was performed using OsMYB86-GFP plants (Supplementary Fig. S16). Two biological ChIP-seq repeats were conducted, resulting in coenrichment of 1,028 genes associated with the binding site. Among these, we found that 3 putative OsMYB86 target genes, BZR1, BU1, and GRF4, were related to the BR signaling pathway (Figs. 5A and S17). To confirm this finding, expression levels of BZR1, BU1, and GRF4 were analyzed in WT plants, and OsMYB86-OE and osmyb86-3 mutant plants. BU1 exhibited higher expression in the lamina joint of OsMYB86-OE plants, but lower expression in that of osmyb86-3 mutant plants, compared with WT (Figs. 5B and S17). BU1 expression was induced efficiently by 2,4-epiBL in WT seedlings, and more efficiently in OsMYB86-OE seedlings, but not in osmyb86-3 mutant seedlings (Fig. 5C), suggesting that BU1 may be the direct target gene regulated by OsMYB86. ChIP-qPCR experiments were performed to test this hypothesis, and substantial enrichment of the P1 and P2 segments of the BU1 promoter sequence by OsMYB86 was evident (Fig. 5, D and E). Notably, both segments contain the core binding sequence [C/T]NGTT[G/T] recognized by R2R3-MYB family proteins (Millard et al. 2019). An electrophoretic mobility shift assay (EMSA) confirmed that OsMYB86-MBP binds directly to segment P1 of the BU1 promoter (Fig. 5F). Consistent with a previous report (Tanaka et al. 2009), the BU1 loss-of-function mutant exhibited an erect leaf phenotype, whereas BU1‐overexpressing plants had increased leaf inclination compared with WT (Figs. 5, G to J and S18). As expected, the erect leaf phenotype of osmyb86-3 mutant plants was substantially suppressed by BU1 overexpression (Fig. 5, G and H), and the enlarged leaf inclination phenotype of OsMYB86-OE plants was substantially suppressed by loss of BU1 function (Fig. 5, I and J). Collectively these results indicate that OsMYB86 likely functions upstream of BU1 in the BR signaling pathway by directly binding to the BU1 promoter, activating its expression.

Figure 5.

OsMYB86 directly promotes BU1 expression in rice. A) Overview of the number of genes associated with OsMYB86 binding sites in 2 ChIP-sequencing replicates. rep, replicate. B) Relative expression level of BU1 in the lamina joint from 60-d-old Kitaake, OsMYB86-OE-1 and osmyb86-3. Asterisks indicate significant difference compared with Kitaake. Data = means ± SD (n = 3, **P < 0.01, Student's t-test). C) Relative expression changes folds of BU1 in the lamina joint from 14-d-old Kitaake, OsMYB86-OE-1 and osmyb86-3 under 1 μM BL treatment. 0, 3, 6, 18, and 24 h represent the treated time by BL. Data = means ± SD (n = 3). D) Diagram of the BU1 promoter region. Asterisks: R2R3-MYB family transcription factors recognize elements ([C/T]NGTT[G/T]). E) ChIP-qPCR assays showing in vivo binding of OsMYB86 to the BU1 promoter. Cross-linked chromatin samples were extracted from Pro35S:OsMYB86-GFP transgenic plants and then precipitated with anti-GFP antibody. Nb (No antibody) served as a negative control. Asterisks indicate significant differences as determined by Tukey's multiple comparisons test Data = means ± SD (n = 3, **P < 0.01, *P < 0.05). F) An EMSA shows that OsMYB86 binds directly to the [C/T]NGTT[G/T] motif in the BU1 promoter. MBP protein, the negative control. The plus (+) and minus (−) signs denote the presence or absence of the protein and DNA probe in each sample. G) Plant phenotypes of Kitaake, BU1-OE, osmyb86-3 and osmyb86-3/BU1-OE at mature stage. Bar = 15 cm. H) Phenotypes and measurements of the flag leaf inclination of Kitaake, BU1-OE, osmyb86-3 and osmyb86-3/BU1-OE at mature stage. Asterisks indicate significant difference compared with Kitaake. Data = means ± SD (n = 10, **P < 0.01, Student's t-test). Bar = 4 cm (applies to all images in Panel H). I) Plant phenotypes of Kitaake, OsMYB86-OE, bu1 and OsMYB86-OE/bu1 at mature stage. Bar = 15 cm. J) Phenotypes and measurements of the flag leaf inclination of Kitaake, OsMYB86-OE, bu1 and OsMYB86-OE/bu1 at mature stage. Asterisks indicate significant difference compared with Kitaake. ns indicates no significance compared with Kitaake. Data = means ± SD (n = 10, **P < 0.01, Student's t-test). Bar = 4 cm (applies to all images in Panel J).

DEP1 acts upstream of OsMYB86 to boost OsMYB86-activated expression of BU1

Both DEP1 and OsMYB86 are positive regulators of BR signaling, and they exhibit physical interaction. To investigate the genetic relationship between them a DEP1-OE/osmyb86-3 hybrid was generated by crossing DEP1-OE and osmyb86-3 plants, and a dep1-1/OsMYB86-OE hybrid was generated by crossing dep1-1 and OsMYB86-OE plants. The flag leaf inclination of DEP1-OE/osmyb86-3 plants was closer to osmyb86-3 plants compared with DEP1-OE plants (Fig. 6, A and B), indicating that DEP1's role in regulating rice leaf inclination is dependent on OsMYB86. In contrast, the flag leaf inclination of dep1-1/OsMYB86-OE plants was closer to that of OsMYB86-OE plants compared with dep1-1 plants (Fig. 6, C and D), suggesting that OsMYB86 overexpression can complement the reduced leaf inclination phenotype caused by loss of DEP1 function. These genetic results demonstrate that OsMYB86 functions downstream of DEP1 in the regulation of rice flag leaf inclination.

Figure 6.

DEP1 acts upstream of OsMYB86 to boost OsMYB86 activated BU1 transcription. A) Plant phenotypes of Kitaake, DEP1-OE, osmyb86-3 and DEP1-OE/osmyb86-3 at mature stage. Bar = 15 cm. B) Phenotypes and measurements of the flag leaf inclination of Kitaake, DEP1-OE, osmyb86-3, and DEP1-OE/osmyb86-3 at mature stage. Asterisks indicate significant difference compared with Kitaake. ns indicates no significance compared with Kitaake. Data = means ± SD (n = 10, **P < 0.01, Student's t-test). Bar = 4 cm (applies to all images in Panel B). C) Plant phenotypes of Kitaake, dep1-1, OsMYB86-OE and dep1-1/OsMYB86-OE at mature stage. Bar = 15 cm. D) Phenotypes and measurements of the flag leaf inclination of Kitaake, dep1-1, OsMYB86-OE and dep1-1/OsMYB86-OE at mature stage. Asterisks indicate significant difference compared with Kitaake. Data = means ± SD (n = 10, **P < 0.01, Student's t-test). Bar = 4 cm (applies to all images in Panel D). E) Effector and reporter constructs used in the dual luciferase assay. F) Representative of dual-luciferase reporter assay co-expressing in N. benthamiana. Co-expressing of ProBU1:luc-Pro35S:Rluc and Pro35S:FLAG and Pro35S:GFP are used as the control (Mock). Renilla luciferase (REN) is used as an internal control. The ratio of LUC/REN represents the relative activity of promoters. The different letters above the histogram indicate significant differences (P < 0.05) by one-way ANOVA followed by Tukey's multiple comparison test. Data= means ± SD (n = 4). G) Relative expression level of BU1 in the lamina joint of Kitaake and gna transgenic lines. Asterisks indicate significant difference compared with Kitaake. Data = means ± SD (n = 3, **P < 0.01, Student's t-test). H) Relative expression level of BU1 in the lamina joint from 60-d-old Kitaake and dep1-1 transgenic lines. Asterisks indicate significant difference compared with Kitaake. Data = means ± SD (n = 3, **P < 0.01, Student's t-test). I) Relative expression changes folds of BU1 in the lamina joint from 16-d-old Kitaake, and dep1-1 under 1 μM BL treatment. 0, 3, 6, 9 and 12 h represent the treated time by BL. Data = means ± SD (n = 3). J) An EMSA shows that DEP1 and GNA do not enhance the binding activity of OsMYB86 to the BU1 promoter. GST and MBP proteins were used as negative controls. The plus (+) and minus (−) signs denote the presence or absence of the protein and DNA probe in each sample.

As mentioned above, OsMYB86 positively regulates BR signaling by directly activating BU1 expression. RNA-seq analysis revealed that dep1, bu1, and gna share a number of differentially expressed genes (DEGs) (Supplementary Fig. S19A), and most of the shared DEGs in dep1, bu1, and gna were changed in the same manner (Supplementary Fig. S19, B to D), suggesting that DEP1, GNA, and BU1 may share a common transcriptional module to regulate rice leaf inclination. In quantitative transactivation assays, co-transfection of ProBU1:LUC and Pro35S:DEP1-FLAG or ProBU1:LUC and Pro35S:DLT2-FLAG did not increase LUC activity. Co-transfection of ProBU1:LUC and Pro35S:OsMYB86-GFP and Pro35S:DEP1-FLAG resulted in a higher LUC activity compared with co-transfection with ProBU1:LUC and Pro35S:OsMYB86-GFP (Fig. 6, E and F). These results suggest that DEP1 enhances the transcriptional activation of BU1 by OsMYB86. Co-transfection of ProBU1:LUC and Pro35S:OsMYB86-GFP and Pro35S:DEP1-FLAG and Pro35S:GNA-FLAG, followed by BL treatment, resulted in the highest LUC activity (Fig. 6, E and F), which is consistent with the strongest nuclear localization signals of DEP1 conferred by BL and GNA (Supplementary Fig. S6, D to F), suggesting that enhanced nuclear entry of DEP1 promotes the transcription of BU1. Supporting this, in RT-qPCR analysis BU1 expression was reduced in dep1-1 and gna mutants compared with WT plants (Fig. 6, G and H). Similar to osmyb86-3 mutants, BL-induced upregulation of BU1 expression was also substantially suppressed in dep1-1 mutants (Fig. 6I). In EMSAs, MBP-OsMYB86 but not MBP-DEP1 or GST-GNA fusion proteins were able to directly bind to BU1-probe. Moreover, DEP1 or GNA did not enhance the binding ability of OsMYB86 to the BU1 promoter (Fig. 6J). Taken together, these results suggest that DEP1 augments the OsMYB86-mediated transcriptional activation of BU1 with the help of GNA, likely by facilitating OsMYB86's function rather than by increasing its binding affinity to the BU1 promoter.

Mutants with loss of Gα (RGA1) function are insensitive to BL (Wang et al. 2006). DEP1 interacts with RGA1, and its role in regulating grain size is dependent on it (Sun et al. 2018). BU1 is a primary BR signaling response gene that functions via both OsBRI1 and RGA1 (Tanaka et al. 2009). Interestingly, the enlarged leaf inclination phenotype of the OsMYB86-OE line was significantly suppressed by the loss of RGA1 function (Supplementary Fig. S20). These findings indicate that the function of OsMYB86 in the BR signaling pathway is also dependent on RGA1, the crucial G‐protein signaling switch.

BU1 interacts with and functions upstream of IBH1 and ILI1

BU1 is a putative non-DNA-binding HLH protein, thus it may function by interacting with other bHLH proteins (Tanaka et al. 2009). To test this hypothesis a yeast 2‐hybrid assay was performed using pGBKT7-BU1 and proteins of the bHLH family known to be involved in BR signaling in rice. BU1 interacted with ILI1 or IBH1 in yeast cells (Fig. 7A). LCI assays further confirmed that BU1 interacted with ILI1 or IBH1 (Fig. 7, B and C). ILI1 and IBH1 are both (b)HLH proteins, and reportedly antagonistically regulate BR signaling and leaf inclination in rice (Zhang et al. 2009a). To investigate how BU1 regulates BR signaling via ILI1 and IBH1, ILI1 and IBH1 knockout Kitaake lines were generated. Consistent with previous reports (Zhang et al. 2009a), the leaf inclination of ILI1 knockout plants (ili1) is smaller than that of the WT, while the leaf inclination of IBH1 knockout plants (ibh1) is larger (Figs. 7, D to G and S21). To further investigate genetic relationships between BU1 and IBH1, or BU1 and ILI1, bu1/ibh1 and bu1/ili1 double mutants were generated. Compared with WT, the flag leaf inclination of both bu1/ibh1 and bu1/ili1 double mutants was close to that of ibh1 and ili1 single mutants (Fig. 7, D to G). These results indicate that BU1 likely functions genetically upstream of ILI1 and IBH1 in the same genetic pathway in the regulation of BR signaling in rice.

Figure 7.

BU1 interacts with and functions upstream of IBH1 and ILI1. A) BU1 interacts with IBH1 and ILI1 in yeast cells. SD-Leu-Trp, selective medium lacking Leu and Trp. SD-Leu-Trp-Ade-His, lacking Trp, Leu, His, and Ade. pGADT7-DLT and pGBKT7-DLT were used as the negative control. B and C) LCI assays verify that BU1 interacts with IBH1 (B) or ILI1 (C) in the leaf epidermal cells of N. benthamiana. DLT-nLUC and DLT-cLUC were used as the negative control. ILI1-nLUC and IBH1- cLUC were used as the positive control. Colored scale bar indicates the luminescence intensity in CPS. D) Plant phenotypes of Kitaake, bu1, ibh1, and bu1/ibh1 at mature stage. Bar = 15 cm. E) Phenotypes and measurements of the flag leaf inclination of Kitaake, bu1, ibh1, and bu1/ibh1 at mature stage. Asterisks indicate significant difference compared with Kitaake. Data = means ± SD (n = 10, **P < 0.01, Student's t-test). Bar = 4 cm. (applies to all images in Panel E). F) Plant phenotypes of Kitaake, bu1, ili1, and bu1/ili1 at mature stage. Bar = 15 cm. G) Phenotypes and measurements of the flag leaf inclination of Kitaake, bu1, ili1, and bu1/ili1 at mature stage. Asterisks indicate significant difference compared with Kitaake. Data = means ± SD (n = 10, **P < 0.01, Student's t-test). Bar = 4 cm. (applies to all images in Panel G). Figure 7, E and G presents statistical data from the same year.

BU1 and ILI1 synergistically relieve IBH1-repressed expression of OsARF11

To further investigate how BU1, ILI1, and IBH1 jointly regulate BR signaling, we aimed to identify a common target gene regulated by these factors. DAP-seq analysis was performed using IBH1-His proteins. Two replicates were conducted, and coenrichment of the OsARF11 promoter region was observed in both samples (Supplementary Fig. S22A). And IBH1 mainly bound to a motif containing GA-repeats in the OsARF11 promoter region (Supplementary Fig. S22B). ChIP-qPCR and EMSAs further confirmed direct binding of IBH1 to the promoter of OsARF11 (Fig. 8, A and B). OsARF11 is the rice homolog of Arabidopsis ARF5/MONOPTEROS, and loss of its function results in reductions in the root system, panicle branches, and grains, and an erect leaf phenotype. Besides its role in auxin signaling, OsARF11 also positively regulates the BR signaling pathway by directly activating BRI1 expression (Sakamoto et al. 2013; Dastidar et al. 2019; Sims et al. 2021). To confirm this, 2 OsARF11 mutants (osarf11-1,2) of Kitaake were generated (Supplementary Fig. S23F), and compared with WT plants both mutants exhibited significantly reduced flag leaf inclination (Supplementary Fig. S23, A and B), and reduced sensitivity to BL treatment (Supplementary Fig. S23, C and D). OsARF11 expression was rhythmically induced by BL, exhibiting 2 peaks of high expression at 0.5 and 9 h during BL treatment (Supplementary Fig. S23E). These findings suggest that OsARF11 might be the downstream gene co-regulated by IBH1, BU1, and ILI1.

Figure 8.

BU1 and ILI1 synergistically relieve the IBH1-repressed transcription of OsARF11.  A) ChIP-qPCR assays showing in vivo binding of IBH1 to the OsARF11 promoter. Nb (No antibody) served as a negative control. Data = means ± SD; asterisks indicate significant differences as determined by Tukey's multiple comparisons test (n = 3, **P < 0.01, *P < 0.05). B) An EMSA shows that IBH1 binds directly to the GA-repeats motif in the OsARF11 promoter. GST protein, the negative control. The plus (+) and minus (−) signs denote the presence or absence of the protein and DNA probe in each sample. C) Effector and reporter constructs used in the dual luciferase assay. IBH1-GFP, BU1-FLAG, and ILI1-FLAG were used as effectors, and GFP and 3 × FLAG as control. A 2,500-bp fragment upstream from the start codon of OsARF11 was fused to LUC as the reporter. D) Representative of dual-luciferase reporter assay in rice protoplasts co-expressing ProOsARF11-min35S:luc-Pro35S:Rluc and Pro35S:IBH1-GFP or ProOsARF11-min35S:luc-Pro35S:Rluc and Pro35S:IBH1-GFP and Pro35S:BU1-FLAG or ProOsARF11-min35S:luc-Pro35S:Rluc and Pro35S:IBH1-GFP and Pro35S:ILI1-FLAG or ProOsARF11-min35S:luc-Pro35S:Rluc and Pro35S:IBH1-GFP and Pro35S:ILI1-FLAG and Pro35S:BU1-FLAG. Co-expressing of ProOsARF11-min35S:luc-Pro35S:Rluc and Pro35S:FLAG and Pro35S:GFP is used as the control (Mock). REN is used as an internal control. The ratio of LUC/REN represents the relative activity of promoters. ns indicates no significance. Data = means ± SD (n = 3). The different letters above the histogram indicate significant differences (P < 0.05) by Tukey's multiple comparison test. E) An EMSA shows that BU1 and ILI1 relieve the binding ability of IBH1 to the OsARF11 promoter. GST protein, the negative control. The plus (+) and minus (−) signs denote the presence or absence of the protein and DNA probe in each sample. F) Relative expression level of OsARF11 in the shoot from Kitaake, ibh1-1 and ibh1-2 mutants at seedling stage. Asterisks indicate significant difference compared with Kitaake. Data = means ± SD (n = 3, **P < 0.01, Student's t-test). G) Relative expression level of OsARF11 in the lamina joint from Kitaake, bu1, ili1, single mutants and bu1/ili1 double mutants at seedling stage. Asterisks indicate significant difference compared with Kitaake. Data = means ± SD (n = 3, **P < 0.01, Student's t-test). H) Subcellular localization of the BU1-GFP&free FLAG and BU1-GFP&ILI1-FLAG in the leaf epidermal cells of N. benthamiana. D53-mCherry, a nuclear marker. Bar = 50 μm. I) The nuclear and cytoplasmic distribution of BU1-GFP protein co-expressed with FLAG or ILI1-FLAG in N. benthamiana leaves. Histone 3 and Actin were used as the nuclear and cytoplasm markers, respectively. C, cytoplasmic fraction; N, nuclear fraction. J) Subcellular localization of the BU1-GFP and ILI1-FLAG and BU1-GFP and FLAG in Kitaake protoplasts. SLG-mCherry, a nuclear and cytoplasm marker. Bar = 10 μm. K and L) Percentage of BU1-GFP fluorescence signal intensity in nuclear and cytoplasm. K) BU1-GFP co-expressed with FLAG in Kitaake protoplasts. L) BU1-GFP co-expressed with ILI1-FLAG in Kitaake protoplasts. Data = means ± SD (n = 30).

Additional analyses were conducted to investigate the regulatory roles of BU1, ILI1, and IBH1 in the modulation of OsARF11 transcription. In quantitative transactivation assays, IBH1-GFP significantly repressed the luciferase activity driven by ProOsARF11-min35S:LUC. This repressive effect was counteracted by BU1-FLAG or ILI1-FLAG, and was more effectively counteracted by the combination of BU1-FLAG and ILI1-FLAG (Fig. 8, C and D). EMSAs indicated that IBH1—but not ILI1 or BU1—could bind to the promoter of OsARF11, and BU1 or ILI1 alone slightly, and BU1 and ILI1 together further inhibited IBH1 binding to the OsARF11 promoter (Fig. 8E). OsARF11 expression levels were upregulated in ibh1-1 and ibh1-2 mutants, but downregulated in bu1 and ili1 single mutants, and greater downregulation was evident in bu1/ili1 double mutants (Fig. 8, F and G). These results indicate a synergistic effect of BU1 and ILI1 in antagonizing the IBH1-repressed transcription of OsARF11.

To test whether ILI1, IBH1, and BU1 could form a heterotrimer to enhance their interaction, an in vitro pull-down assay was performed. The addition of increasing amounts of ILI1-GST did not enhance interaction between IBH1 and BU1 (Supplementary Fig. S24). In a previous report fluorescence of eGFP-BU1 was mainly distributed in the cytoplasm of rice coleoptile cells (Tanaka et al. 2009), thus we repeated the experiment in Kitaake protoplasts. Consistent with that previous report BU1-GFP fluorescence was mainly distributed in the cytoplasm with a relatively weak nuclear localization signal, whereas IBH1-GFP was localized in the nucleus, and ILI1-GFP was localized in both the nucleus and the cytoplasm (Supplementary Fig. S25A). Similar subcellular localization patterns of ILI1-GFP, IBH1-GFP, and BU1-GFP were also observed in leaf epidermal cells of N. benthamiana (Supplementary Fig. S25A). Interestingly, when BU1-GFP and ILI1-FLAG proteins were co-expressed in the epidermal cells of N. benthamiana leaves, there was an increased nuclear BU1-GFP fluorescence signal (Fig. 8H). Consistent with this, in western blotting analysis the BU1-GFP band was faint without ILI1, but became distinct in the presence of ILI1 in the cell nucleus of N. benthamiana leaves (Fig. 8I). Similarly, the subcellular localization signal of BU1-GFP alone was mainly distributed in the cytoplasm of Kitaake or ili1 protoplasts (Figs. 8J and S25B), and co-expression of BU1-GFP and ILI1-FLAG in Kitaake protoplasts also increased nuclear BU1-GFP fluorescence signals (Fig. 8, K and L). These findings indicate that the presence of ILI1 facilitates the translocation of BU1 from the cytoplasm to the nucleus.

Discussion

In animals and plants, heterotrimeric G-proteins transmit extracellular signals into intracellular signaling components (Gilman 1987; Urano et al. 2013). However, the precise interplay between G-protein components and BR signaling in rice remains largely unexplored. Herein we report that the DEP1-GNA-OsMYB86- BU1/ILI1/IBH1-OsARF11 pathway links the Gγ subunit DEP1 to the BR signaling pathway. First, we found that DEP1 transgenic plants exhibited BR-related phenotypes, while dep1 mutants showed reduced BR sensitivity, and DEP1-OE plants exhibited increased BR sensitivity (Fig. 1). Second, BRs facilitated the nuclear import of DEP1 via the BR signaling protein GNA (Figs. 2 and S6). Third, we showed that DEP1 interacts with OsMYB86, a MYB transcription factor that positively regulates BR signaling, and that both are in the same genetic pathway that regulates leaf inclination in rice (Figs. 3 to 6). Fourth, we revealed that DEP1 boosts the OsMYB86-promoted transcription of BU1 with the help of GNA (Fig. 6). Fifth, we found that by interacting with BU1, ILI1 promotes the importation of BU1 into the nucleus, where they synergistically and efficiently relieve the IBH1-repressed transcription of OsARF11 (Figs. 7 and 8), a gene involved in both auxin and BR signaling. Lastly, we found that the function of the DEP1-OsMYB86-BU1 pathway in BR signaling is likely dependent on RGA1 (Supplementary Fig. S20) (Wang et al. 2006; Tanaka et al. 2009; Sun et al. 2018). However, the precise mechanism underlying RGA1's regulation in the DEP1-OsMYB86-BU1 pathway remains unclear and requires further research.

Recently, we found that GNA interacts with DEP1 and facilitates its entry into the cell nucleus (Zhang et al. 2024). Here, we further confirmed that in the presence of GNA and BR, the nuclear entry of DEP1 proteins was more effectively promoted (Figs. 2 and S6). Notably, GNA is a GRAS family protein that has been shown to possess an innate capability to activate RNA polymerase (Hirsch et al. 2009). Therefore, upon entering the cell nucleus, DEP1 interacts with BR signaling-related transcription factors such as OsMYB86 (Fig. 3) and GNA (Zhang et al. 2024), potentially forming a transcriptional regulatory complex to efficiently promote the transcription of a key downstream gene BU1 (Fig. 6F). Because we were unable to detect DEP1 through Western blot analysis in 35S:DEP1-GFP transgenic plants, as observed in both our study and previous reports (Taguchi-Shiobara et al. 2011; Sun et al. 2018), our conclusions that BRs promote DEP1's nuclear localization are primarily based on transient expression systems. This limits further investigation into the roles of the rice G-protein complex in planta. Therefore, identifying the underlying reasons for the undetectable DEP1 in transgenic plants is valuable for a comprehensive understanding of the detailed roles of DEP1 in rice plants.

The bHLH superfamily is a transcription factor family containing many members, and it is widely found in both plants and animals (Hao et al. 2021). Many members of the bHLH family have been implicated in BR signaling in rice, including BU1 (Tanaka et al. 2009), ILI1, IBH1 (Zhang et al. 2009a), OsBUL1 (Jang et al. 2017), OsbHLH98 (Guo et al. 2021), and OsbHLH92 (Teng et al. 2023). Based on the phylogenetic relationship and DNA motif binding capacities of bHLH family members, 6 major groups have been identified within the bHLH family. IBH1 is not included in any of the 6 major groups, however, and its DNA-binding capacity and preferred binding motif are unknown (Hao et al. 2021). In addition, although we discover a mechanism by which DEP1, GNA and OsMYB86 activate the expression of BU1, how BU1 together with ILI1 and IBH1 transmits BR signal remains unclear. By further investigations, we found that IBH1 directly repressed the expression of OsARF11, a gene involved in BR and auxin crosstalk (Sakamoto et al. 2013), by binding to the GA-repeat sequences in the OsARF11 promoter. With respect to the specific mechanism by which they regulate BR signaling, ILI1 likely facilitates the importation of BU1 into the nucleus where together they synergistically and efficiently inhibit binding of IBH1 to the promoter of OsARF11, thus attenuating the IBH1‐repressed transcription of OsARF11, finally promoting BR signaling (Figs. 8, S22, S24, and S25). These results enhance our understanding of the regulatory mechanism of BR signaling by HLH/bHLH proteins.

As well as BR, auxin is an important hormone that regulates rice leaf inclination, and the genes involved in the auxin synthesis and signaling pathway affect rice leaf inclination (Zhang et al. 2009b; Bian et al. 2012; Huang et al. 2021). Among the auxin-related genes affecting rice leaf inclination, auxin-responsive factors (ARFs) have been extensively reported to regulate rice leaf inclination by mediating crosstalk between auxin and BR. For example, some ARFs such as OsARF1 and OsARF4 act as negative regulators of BR signaling, inhibiting rice leaf inclination by mediating crosstalk between auxin and BR (Song et al. 2009; Qiao et al. 2022). In contrast, other ARFs such as OsARF11 and OsARF19 positively regulate rice leaf inclination by promoting the transmission of BR signaling (Sakamoto et al. 2013; Zhang et al. 2015b). In the present study BU1, ILI1, and IBH1 converged and directly controlled OsARF11 expression (Fig. 8), which provides insights into the key role of auxin in regulating leaf inclination and crosstalk between auxin and BR.

Based on the above findings, we propose the following working model (Fig. 9). RGA1, acting as a key signaling switch, triggers BR signaling by activating the DEP1-mediated pathway in rice. As BR levels increase in the plant, BRs promote the accumulation of GNA proteins, which in turn facilitate the nuclear entry of DEP1. In the nucleus, DEP1 interacts with OsMYB86 and GNA, likely forming a transcriptional regulatory complex to effectively activate OsMYB86-mediated transcription of BU1. Additionally, BRs promote the expression of the BR positive response gene ILI1, while repressing the BR negative response gene IBH1. The ILI1 protein then interacts with BU1, facilitating the transfer of BU1 from the cytoplasm to the nucleus, where together they inhibit IBH1's binding to the promoter of OsARF11. This relieves IBH1-mediated repression of OsARF11 transcription, thereby activating BR responses and increasing leaf inclination in rice. These findings suggest that the BR signaling pathway mediated by DEP1 effectively activates BR responses in rice. This offers insights into the mechanisms of BR signaling and provides a theoretical basis for breeding rice cultivars with optimal leaf inclination for dense planting.

Figure 9.

A proposed working model for the activation of BR responses by DEP1-mediated signaling pathways. As BR levels increase in the plant, the nuclear localization of DEP1 is enhanced with the help of GNA. In the nucleus, DEP1, GNA, and OsMYB86 likely form a complex to enhance OsMYB86's transcriptional activation of BU1. At the same time, BRs also upregulate the expression of ILI1 while suppressing IBH1 expression. The ILI1 protein then interacts with BU1, promoting its nuclear import, where both synergistically relieve the IBH1-mediated repression of OsARF11 transcription, ultimately activating BR responses and increasing leaf inclination.

Materials and methods

Plants and growth conditions

To generate dep1, osmyb86, d1, bu1, ili1, ibh1, and osarf11 knockout plants, 20-bp gene-specific spacer sequences of DEP1, OsMYB86, RGA1, BU1, ILI1, IBH1, and OsARF11 were inserted into the sgRNA/Cas9 construct, respectively (Miao et al. 2013). To generate a DEP1 overexpression construct, the full-length coding sequence of DEP1 was cloned into the binary vector pCAMBIA1305GFP to produce Pro35S:DEP1-GFP. The full-length coding sequence of OsMYB86 was amplified and cloned into the binary vectors pCUbi1390, pCAMBIA2300, and pCAMBIA1305 to produce OsMYB86 overexpression constructs ProUbi:OsMYB86, ProActin:OsMYB86, and Pro35S:OsMYB86-GFP. The above constructs were introduced into Agrobacterium tumefaciens strain EHA105, then transformed into the callus of the japonica cultivar variety, Kitaake. To obtain OsMYB86-OE/bu1 plants the callus of Kitaake was transformed with a mix of A. tumefaciens containing ProActin:OsMYB86 and bu1-sgRNA/Cas9 constructs. ProUbi:OsMYB86 and bu1 plants were crossed to obtain OsMYB86-OE/bu1 plants. ProActin:BU1 and osmyb86-3 plants were crossed to obtain BU1-OE/osmyb86-3 plants. Pro35S:DEP1-GFP and osmyb86-3 plants were crossed to obtain DEP1-OE/osmyb86-3 plants. ProUbi:OsMYB86 and dep1-1 plants were crossed to obtain OsMYB86-OE/dep1-1 plants. ProUbi:OsMYB86 and d1 plants were crossed to obtain OsMYB86-OE/d1 plants. bu1 and ibh1 plants were crossed to obtain bu1/ibh1 plants bu1 and ili1 plants were crossed to obtain bu1/ili1 plants. All plants were grown in the experimental field of the Chinese Academy of Agricultural Sciences under natural conditions with conventional management. The detailed primer information is provided in the Supplementary Table S1.

Subcellular localization

For subcellular localization of DEP1, OsMYB86, BU1, ILI1, and IBH1 protein, the full-length coding sequences of DEP1, OsMYB86, BU1, ILI1, and IBH1 were amplified and cloned into the transient expression vector pAN580 to generate Pro35S:DEP1/OsMYB86/BU1/ILI1/IBH1-GFP fusion plasmids. The Pro35S:DEP1/OsMYB86/BU1/ILI1/IBH1-GFP fusion plasmids were transformed into rice protoplasts as previously described (Zhang et al. 2011). After incubation at 25 °C for 6 to 16 h, fluorescence detection was performed. Full-length coding sequences of DEP1, OsMYB86, BU1, IBH1, and ILI1 were cloned into pCAMBIA1305 to generate Pro35S:DEP1/OsMYB86/BU1/ILI1/IBH1-GFP fusion plasmids. The vectors were introduced into A. tumefaciens strain EHA105, then N. benthamiana leaves were exposed to specific combinations. Fluorescent signals were monitored 48 to 72 h after exposure. p35S:D53-mCherry was used as a nucleus marker (Zhou et al. 2013), Pro35S:SLG-mCherry was used as a nucleus and cytoplasm marker (Feng et al. 2016). Fluorescence signals were observed via a Zeiss LSM980 confocal microscope.

Subcellular localization analysis of DEP1

To investigate the effects of BL on DEP1-GFP localization in N. benthamiana leaf epidermal cells, a final concentration of 2 μM of 2,4-epiBL mixed with Agrobacterium-containing mediator solution was used for infiltration into N. benthamiana leaves. The same volume of ethanol mixed with Agrobacterium-containing mediator solution was used as a control. Fluorescent signals were monitored 60 to 72 h after infiltration.

Subcellular localization analysis of BU1

To investigate the effects of ILI1 on BU1 localization Kitaake protoplasts were transformed with Pro35S:BU1-GFP with free FLAG and Pro35S:ILI1-FLAG, respectively, or ili1 protoplasts were transformed with free FLAG. Fluorescent signals were monitored 48 to 72 h after infiltration. The signal intensity of BU1-GFP in the nucleus and the signal intensity of the entire cell were quantified via ZEN 3.1 (blue edition) to calculate the percentage of BU1 signal in the nucleus.

Yeast 2-hybrid assay

The coding region of DEP1 was fused to the GAL4 binding domain of the pGBKT7 vector as a bait. A cDNA library from young rice inflorescences was used to perform Y2H screening, and positive clones were identified via sequencing. Full-length coding sequences of RGB1, RGA1, GGC2, GS3, and BU1 and various truncated versions of DEP1 were cloned into pGBKT7. Full-length coding sequences of OsMYB86, ILI1, and IBH1 were cloned into pGADT7. pGADT7-DLT, pGBKT7-DLT, and pGBKT7-RGA1 were used as negative controls. Various combinations of plasmids were cotransformed into the yeast strain AH109 (Clontech). After growing on SD-Trp/-Leu plates for 3 d at 30 °C, interactions were observed on the selective medium SD-Leu/-Trp/-His/-Ade.

BiFC assay

Full-length coding sequences of OsMYB86 and DEP1 were fused to p2YC and p2YN vectors, respectively. The plasmids were transformed into A. tumefaciens (strain EHA105) and infiltrated into N. benthamiana leaves as previously described (Waadt and Kudla 2008). P2YN-RGA1 and P2YC-DLT were used as negative controls. Fluorescent signals were monitored 48 to 72 h after infiltration via a Zeiss LSM980 confocal microscope.

LCI assay

Full-length coding sequences of OsMYB86, IBH1, and ILI1 were fused to the pCAMBIA1300-Cluc vector. Full-length coding sequences of DEP1 and BU1 were fused to the pCAMBIA1300-nLUC vector. The vectors were introduced into A. tumefaciens strain EHA105, then infiltrated into N. benthamiana leaves. RGA1-nLUC, DLT-nLUC, and DLT-cLUC were used as negative controls. ILI1-nLUC and IBH1‐cLUC were used as positive controls. After 36 to 48 h, N. benthamiana leaves were treated with 1 mm luciferin (E1601, Promega) for 3 min, then the luciferase activities were measured using an imaging apparatus (LB 985, Berthold).

ChIP-seq and ChIP-qPCR

ChIP-seq assays were conducted by SeqHealth (Wuhan, China) using the leaves, stems, and lamina joints of Pro35S:OsMYB86-GFP transgenic plants, with 2 biological replicates. Anti-GFP antibodies (598-7, Medical Biological Laboratories) were used, and the sequencing depth was set at 20 million reads per sample. Raw sequencing data were filtered using Trimmomatic (version 0.36) to remove low-quality reads and trim adapter sequences. Read distribution analysis was performed with RSeQC (version 2.6), and peak calling was conducted using MACS2 (version 2.1.1). Peak annotation and distribution analysis were performed using bedtools (version 2.25.0). Differential binding peaks were identified via a Fisher's test using a custom Python script. Motif analysis was conducted using Homer (version 4.10). GO and Kyoto Encyclopedia of Genes and Genomes enrichment analyses for annotated genes were performed using KOBAS (version 2.1.1), with a corrected P value threshold of 0.05 to determine statistically significant enrichment. ChIP-qPCR assays were performed as previously described (Wang et al. 2018). Leaves, stems, and lamina joints of Pro35S:OsMYB86-GFP transgenic plants or young N. benthamiana leaves cotransformed with the Pro35S:OsMYB86-GFP and ProBU1:LUC vectors were used to test the enrichment of OsMYB86 at the promoter regions of BU1. Young N. benthamiana leaves cotransfected with Pro35S:IBH1-GFP and ProOsARF11:LUC vectors were used to test the enrichment of IBH1 in the promoter regions of OsARF11. GFP antibodies (Medical Biological Laboratories, 598) were used for detection.

LUC activity assay

An approximately 2.5-kb promoter region of BU1 and OsARF11 was cloned to fuse into the pGreenII0800-LUC vector to generate ProBU1:LUC and ProOsARF11:LUC reporters. Full-length coding sequences of OsMYB86 and IBH1 were cloned to fuse into the pCAMBIA1305GFP vector to generate Pro35S:OsMYB86-GFP and Pro35S:IBH1‐GFP effectors. Full-length coding sequences of DEP1, GNA, ILI1, and BU1 were cloned to fuse into the pCAMBIA1300-FLAG vector to generate Pro35S:DEP1-FLAG, Pro35S:GNA-FLAG, Pro35S:ILI1-FLAG, and Pro35S:BU1-FLAG effectors. Empty vectors were used as negative controls. The combined reporter and effector plasmids were cotransformed into rice protoplasts. Various combinations of plasmids were also cotransformed into A. tumefaciens (strain EHA105) then infiltrated into N. benthamiana leaves. LUC activity was quantified with a Dual-Luciferase Assay Kit (Promega) in accordance with the manufacturer's instructions, and relative LUC activity was calculated as the ratio of LUC/REN.

EMSA

To perform EMSAs, full-length coding sequences of OsMYB86 and DEP1 were cloned into the pMAL-c2x vector. Full-length coding sequences of ILI1, IBH1, and BU1 were cloned into pGEX4T-1. The correct constructs of MBP-OsMYB86, MBP-DEP1, ILI1-GST, BU1-GST, IBH1-GST, and empty MBP and GST vectors were introduced into the Escherichia coli strain DE3 to induce protein expression. MBP and MBP‐labeled protein were eluted with 10 mm maltose. GST and GST-labeled protein were eluted with 20 mm glutathione. Oligonucleotide probes were synthesized and labeled with biotin by Thermo Fisher Scientific. EMSA was then performed using the lightshift Chemiluminescent EMSA Kit (Thermo, 20148).

RNA extraction and RT-qPCR analysis

Total RNA was extracted from lamina joints of 70-d-old plants and 2-wk-old BL treatment seedlings using the ZR Plant RNA MiniPrep Kit (Zymo Research) in accordance with the manufacturer's instructions. Total RNAs were reverse transcribed using a Reverse Transcription Kit (Qiagen). RT-qPCR analyses were performed using an ABI 7500 realtime PCR system with a SYBR Premix Ex Taq II Kit (Takara). The rice Ubiquitin (UBQ) gene was used as an internal control.

Co-IP assay

To detect OsMYB86-DEP1 interaction in vivo, full-length coding sequences of DEP1 and OsMYB86 were cloned into pCAMBIA1305.1-GFP and pCAMBIA1300-FLAG vectors, respectively. The plasmids were cotransformed into A. tumefaciens (strain EHA105) then infiltrated into N. benthamiana leaves. After 48 h treatment, total protein was extracted from infiltrated N. benthamiana leaves. Anti-GFP (598-7, Medical Biological Laboratories, 1:5000) and anti-FLAG antibodies (M185-7, Medical Biological Laboratories, 1:5000) were used in immunoblotting (IB) analysis.

BL treatment

BL was dissolved in ethanol. For the lamina inclination test, approximately 14-d-old rice seedlings with expanded third leaves were soaked in rice nutrient solution supplemented with different concentrations of 2,4-epiBL. Ethanol was used as a mock treatment. Images of plants were then taken for lamina inclination measurement at 24 to 48 h after treatment, and lamina inclination was measured using ImageJ software. The coleoptile length test was performed as previously described (Tong and Chu 2017). Seeds were sterilized and germinated on 1% agar medium containing different concentrations of BL. After 7 d of growth at 30 °C the length of coleoptiles was measured.

Flag leaf inclination observation and measurement

After the rice's main panicle had fully emerged, uniform samples were collected by cutting segments that included the panicle, leaf lamina joint, and leaf blade. Photographs were then taken, and blade inclinations were measured using ImageJ software.

Phylogenetic analysis

Gene sequences used in phylogenetic analysis were downloaded from https://phytozome-next.jgi.doe.gov/, and a phylogenetic tree was constructed using MEGA5 software and the neighbor-joining method with 1,000 bootstrap replicates. The sequences used to construct the phylogenetic tree are provided in Supplementary Data Set 2.

In vitro pull-down assay

Full-length coding sequences of IBH1, ILI1, and BU1 were cloned into the expression vectors pET-28a, pGEX4T-1, and pMAL-c2x, respectively, to generate His, GST, and MBP tag fusion proteins. IBH1-His, ILI1-GST, GST, BU1-MBP, and MBP proteins were then expressed in the E. coli strain BL21 (DE3) (TransGen) under induction with 0.5 mm isopropyl-b-D-thiogalactoside, and shaking at 16 °C for 16 h. Fusion proteins were purified using GST magnetic beads (BEAVER), His magnetic beads (BEAVER), or amylose magnetic beads (Biolabs) in accordance with the manufacturer's instructions. To detect BU1-ILI1-IBH1 interaction using the in vitro pull-down assay, approximately equal amounts of GST and GST-ILI1 or MBP and MBP-BU1 were mixed with His-IBH1, then the mixed supernatants were incubated with 30 μL of His magnetic beads in 1.5 mL phosphate-buffered saline. After incubation for 60 min the beads were washed 6 times with phosphate-buffered saline, then boiled with 100 μL protein loading buffer at 100 °C for 10 min. The proteins were separated in 10% SDS‐PAGE gels and detected via western blotting using anti-GST antibody (PM013-7, Medical Biological Laboratories, 1:5000), anti-His antibody (D291-7, Medical Biological Laboratories, 1:5000), and anti-MBP antibody (E8032S, BioLabs, 1:5000).

Fractionation of proteins and IB

Protein fractionation and IB assays for N. benthamiana were performed using a commercial nucleus/cytoplasm separation kit (Beyotime P0028) according to the manufacturer's instructions. For rice protoplast protein fractionation and IB assays, prepare a sufficient amount of rice protoplasts. Centrifuge 250 × g of the sample for 5 min to collect the protoplasts, discard the supernatant, and retain the pellet. Resuspend the pellet in cytoplasmic protein extraction reagent (Beyotime P0028), vortex for 5 s, incubate on ice for 10 min, and centrifuge at 12,000 × g for 10 min at 4 °C. Carefully collect the supernatant to obtain cytoplasmic proteins. Prepare 60% and 30% sucrose solutions using the cytoplasmic protein extraction reagent and slowly layer them sequentially into a centrifuge tube. Resuspend the crude nuclear pellet in the cytoplasmic protein extraction reagent and gently load it onto the top of the sucrose gradient. Centrifuge at 20,000 × g for 2 h at 4 °C. Carefully collect the white interface between the 30% and 60% sucrose layers and resuspend it in nuclear protein extraction reagent (Beyotime P0028) to obtain nuclear proteins. Full-length coding sequences of DEP1 and BU1 were cloned into pCAMBIA1305.1-GFP vector. Full-length coding sequences of GNA and ILI1 were cloned into pCAMBIA1300-FLAG vector. The plasmids were cotransformed into A. tumefaciens (strain EHA105), then infiltrated into N. benthamiana leaves. 60 to 72 h after infiltration, 0.5 g N. benthamiana leaves were harvested for subsequent experiments. Full-length coding sequences of DEP1was amplified and cloned into the transient expression vector pAN580 to generate Pro35S:DEP1-GFP fusion plasmids. The Pro35S:DEP1-GFP fusion plasmids were transformed into rice protoplasts as previously described (Zhang et al. 2011). After incubation at 25 °C for 12 h, protoplasts were harvested for subsequent experiments. Twenty microliters of cytoplasmic or nuclear fractions were used in immunoblot analysis performed with anti-GFP (598-7, Medical Biological Laboratories, 1:5000), anti-H3 (ab1791, abcam, 1:1000), anti-β-actin (BE0033, Easybio, 1:1000), and anti-actin (AC009, ABclonal, 1:1000).

Statistical analysis

The statistical results are indicated as means ± SD, where n represents the number of biological replicates. GraphPad Prism 5.0 was used for statistical analysis. Detailed statistical analysis data are provided as Supplementary Data Set 1.

Accession numbers

Sequences of genes involved in this study can be found in Rice Genome Annotation Project https://rice.uga.edu/, and Phytozome https://phytozome-next.jgi.doe.gov/under the accession numbers LOC_Os09g26999(DEP1), LOC_Os03g51330(GNA), LOC_Os01g50720(OsMYB86), LOC_Os06g12210(BU1), LOC_Os04g54900(ILI1), LOC_Os04g56500(IBH1), LOC_Os05g26890(RGA1), LOC_Os04g56850(OsARF11), LOC_Os06g03710(DLT), LOC_Os03g46650(RGB1), LOC_Os09g32510(OsbHLH92), LOC_Os01g10040(D2), LOC_Os04g39430(D11), LOC_Os03g40540(BRD1), LOC_Os10g25780(BRD2), LOC_Os02g47280(OsGRF4), LOC_Os05g39950(OsOFP22). Sequencing data of ChIP-seq and RNA-seq can be found in NCBI https://www.ncbi.nlm.nih.gov/ (Bioproject, PRJNA1159116, ChIP-seq) (Bioproject, PRJNA1159124, PRJNA1211729, RNA-seq).

Author contributions

J.Wan and Z.C. supervised the project; S.Li and Q.L. designed the research and wrote the paper; Z.C. and Z.Z. performed most of the plant hybridization experiments; T.L. provided the plant material of OsMYB86; J.Z. provided the plant material of GNA. S.Li performed most of the experiments; X.X. prepared the rice protoplasts; X.L. provided technical assistance of the ChIP assay and bioinformatics analysis; M.F., S.Luo, K.D., Y.W., F.Z., J.W., R.M., W.L., C.L., Y.R., S.Z., and X.W. provided technical assistance; Xiuping Guo generated the transgenic plants.

Supplementary data

The following materials are available in the online version of this article.

Supplementary Figure S1. Generation of knockout and overexpression transgenic lines of DEP1.

Supplementary Figure S2. Coleoptile elongation assay of Kitaake and dep1-1 in response to BL.

Supplementary Figure S3. GO enrichment analysis of DEGs in dep1-1 vs WT.

Supplementary Figure S4. Heatmap showing the expression of DEGs related to BR signaling and biosynthesis in Kitaake, DEP1-OE, and dep1-1 mutants.

Supplementary Figure S5. The expression of BR-responsive genes in Kitaake and dep1-1 mutant under BL treatment.

Supplementary Figure S6. BRs promote DEP1 nuclear entry through GNA in N. benthamiana.

Supplementary Figure S7. Yeast screening for interacting proteins of DEP1 and yeast 2-hybridization assay with truncated OsMYB86.

Supplementary Figure S8. Phylogenetic analysis of OsMYB86 and its MYB family homologs.

Supplementary Figure S9. Protein sequence alignment of OsMYB86 and its homologs.

Supplementary Figure S10. Expression level of OsMYB86 in different plant tissues of Kitaake.

Supplementary Figure S11. An in vitro pull-down assay indicates that OsMYB86 does not interact with GNA.

Supplementary Figure S12. Subcellular localization of OsMYB86 and DEP1.

Supplementary Figure S13. OsMYB86 slightly promoted DEP1's nuclear localization.

Supplementary Figure S14. Generation of knockout and overexpression transgenic lines of OsMYB86.

Supplementary Figure S15. Grain width and thickness of Kitaake and OsMYB86 transgenic lines.

Supplementary Figure S16. Detection of OsMYB86-GFP protein in Pro35S:OsMYB86-GFP transgenic lines used for ChIP-Seq and ChIP-qPCR analyses.

Supplementary Figure S17. ChIP-seq analysis of OsMYB86.

Supplementary Figure S18. Generation of knockout and overexpression transgenic lines of BU1.

Supplementary Figure S19. RNA-seq analysis of Kitaake, dep1, gna, and bu1.

Supplementary Figure S20. Functional dependence of OsMYB86 on RGA1.

Supplementary Figure S21. Generation of knockout plants of ILI1 and IBH1.

Supplementary Figure S22. DAP-seq analysis of IBH1.

Supplementary Figure S23.  OsARF11 regulates rice flag leaf inclination by influencing BR signaling.

Supplementary Figure S24. An in vitro pull-down assay shows that ILI1 could not enhance the interaction between BU1 and IBH1.

Supplementary Figure S25. Subcellular localization of ILI1-GFP, IBH1-GFP, and BU1-GFP.

Supplementary Table S1. Primers used in this study.

Supplementary Data Set 1. Summary of statistical tests.

Supplementary Data Set 2. Sequence alignments used for constructing the phylogenetic tree.

Funding

This research was supported by Biological Breeding-National Science and Technology Major Project (2024ZD04080), the Project of National Nanfan Research Institute of CAAS (YBXM2425), the Agricultural Science and Technology Innovation Program (CAAS-ZDRW202401), and the National Key Research and Development Program of China (2022YFF1002900, 2022YFD1200104).

Data availability

The data underlying this article are available within the article and Supplementary files. The plant materials and antibodies during the study are available from the corresponding authors upon reasonable request.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

• RGA1 Gramene: AT2G01570

• RGA1 Araport: AT2G01570

• BZR1 Gramene: AT1G75080

• BZR1 Araport: AT1G75080

• BIN2 Gramene: AT4G18710

• BIN2 Araport: AT4G18710

• BSU1 Gramene: AT1G03445

• BSU1 Araport: AT1G03445

• ILI1 Gramene: Os04g0641700

• ILI1 Araport: Os04g0641700

• BRI1 Gramene: AT4G39400

• BRI1 Araport: AT4G39400

• DEP1 Gramene: Os09g0441900

• DEP1 Araport: Os09g0441900

• RGB1 Gramene: Os03g46650

• RGB1 Araport: Os03g46650

• DLT Gramene: Os06g0127800

• DLT Araport: Os06g0127800

• GGC2 Gramene: Os08g0456600

• GGC2 Araport: Os08g0456600

• NCBI Gramene: https://www.ncbi.nlm.nih.gov

• NCBI Araport: https://www.ncbi.nlm.nih.gov

• PCR Gramene: Polymerase Chain Reaction

• PCR Araport: Polymerase Chain Reaction

• OsBRI1 Gramene: Os01g0718300

• OsBRI1 Araport: Os01g0718300