*2.5. OsbHLH079 Regulates the Expression of BR Signaling-Related Genes*

The wide leaf angle phenotype of *osbhlh079-D* resembles that of mutants with elevated BR accumulation or enhanced BR signaling [11,14,15,61–63]. Moreover, the transcript levels of several XTHs and expansin genes, which are upregulated in *osbhlh079-D* (Figure 5c), are significantly increased by BR treatment in *Arabidopsis thaliana*, rice, soybean (*Glycine max*), maize (*Zea mays*), and wheat (*Triticum aestivum*) [59,64–70]. Therefore, we speculated that the increased leaf angle of *osbhlh079-D* is caused by either elevated endogenous BR accumulation or enhanced BR signaling.

To investigate whether the expression of BR biosynthesis- or BR signaling-related genes is altered in *osbhlh079-D*, we compared their transcript levels in the lamina joints of leaf blades between WT and *osbhlh079-D*. In the lamina joints, the expression of BR biosynthesis-related genes, such as *D2*, *D11*, and *BRD1* [15,16,61], was significantly downregulated in *osbhlh079-D* compared to that of WT (Figure 7a). In addition, the transcript level of *OsBRI1*, the BR receptor, was also significantly downregulated compared to that of WT (Figure 7b), indicating a negative feedback regulation by enhanced BR signaling [14,63,71]. Among the BR signaling-related genes, including *OsBAK1*, *OsBSK3*, *GSK2*, *BU1*, *OsBZR1*, *ILI1*, and *DLT* [14,30,47,48,72–75], the expression of *OsBZR1*, and its downstream genes, such as *ILI1*, and *DLT*, was significantly altered in the lamina joint of *osbhlh079-D* compared to WT (Figure 7b). For example, the expression of genes encoding positive regulators of the BR signaling pathway, such as *OsBZR1*, and *ILI1*, was highly upregulated, but the transcript level of *DLT*, which also encodes a positive regulator of BR signaling pathway but is repressed directly by OsBZR1, was significantly downregulated in *osbhlh079-D* (Figure 7b). To confirm whether the expression levels of *OsBZR1*, *ILI1*, and *DLT* are altered by the ectopic or knockdown expression of *OsbHLH079*, we compared the expression levels of *OsBZR1*, *ILI1*, and *DLT* in the lamina joint among WT, *35S::OsbHLH079*, and *35S::RNAi-OsbHLH079*. The transcript levels of *OsBZR1*, and *ILI1* were highly upregulated, while *DLT* expression was significantly downregulated in the lamina joint of *35S::OsbHLH079* lines, as in *osbhlh079-D* (Figure 7c–e). By contrast, the expression levels of *OsBZR1*, and *ILI1* were significantly decreased, while the transcript level of *DLT* was highly increased in the lamina joint of *35S::RNAi-OsbHLH079* lines (Figure 7c–e). These results indicated that the increased activity of OsbHLH079 enhances the BR signaling pathway by altering the expression of *OsBZR1* and its downstream genes, such as *ILI1*, and *DLT*.

To verify whether the response to BR treatment is enhanced by the overexpression of *OsbHLH079*, we carried out a BR-induced lamina joint inclination assay. For this assay, 2-cm lamina joint segments were detached from 10-day-old seedlings of WT and *osbhlh079-D* grown in darkness and treated with 1 μM BL for 48 h in darkness. Then, we compared the extent of lamina inclination of *osbhlh079-D* with WT. As shown in Figure 7f, *osbhlh079-D* was more sensitive to BR (24-epibrassinolide) treatment. Moreover, the difference in the extents of lamina inclination between WT and *osbhlh079-D* increased as the BR concentration increased (Figure 7b). These data indicated that BR signaling is enhanced in *osbhlh079-D*. Therefore, we concluded that OsbHLH079 enhances the BR signaling pathway, which leads to the expansion of cell size in the adaxial side of lamina joints via upregulation of *OsEXPAs* and *OsXTHs*, resulting in an increase in leaf angle in rice (Figure 8).

**Figure 7.** OsbHLH079 acts as a positive regulator of the brassinosteroid signaling pathway. (**a**) Expression patterns of brassinosteroid (BR) biosynthesis-related genes in the *osbhlh079-D* mutant compared to those in WT. (**b**) Altered expressions of BR signaling-related genes in the *osbhlh079-D* mutant compared to those in WT. (**c**–**e**) Altered expressions of *OsBZR1* (**c**), *ILI1* (D), and *DLT* (E) in *35S::OsbHLH079* and *35S::RNAi-OsbHLH079* compared to those in WT. (**a**–**e**) Total RNA was extracted from the 2-cm lamina joints between leaf blade and leaf sheath of 4-week-old plants of WT, *osbhlh079-D*, *35S::OsbHLH079*, and *35S::RNAi-OsbHLH079* grown under LD conditions (14.5 h light, 30 ◦C/9.5 h dark, 24 ◦C) with 60% relative humidity in a growth chamber. The transcript level of each gene was determined by RT-qPCR analysis and normalized to *UBQ5*. Means and standard deviations were obtained from three biological replicates. Asterisks indicate statistically significant differences (\* *p* < 0.05, \*\* *p* < 0.01, Student's *t*-test) compared to WT. (**f**) BR-induced lamina joint inclination in WT and the *osbhlh079-D* mutant. The 2-cm lamina joint segments of 10-day-old seedlings of WT and *osbhlh079-D* grown at 30 ◦C in darkness were treated with 1 μM BL for 48 h in darkness. Scale bar = 0.5 cm. BL, 24-epibrassinolide. (**g**) Dose-dependent responses of the lamina joint of WT and *osbhlh079-D* to various concentrations of BL. Means and standard deviations were obtained from more than ten biological replicates. Significant differences between means were analyzed using Student's *t*-test (\* *p* < 0.05, \*\* *p* < 0.01). These experiments were repeated twice with similar results. BL, 24-epibrassinolide.

**Figure 8.** A proposed model of the OsbHLH079-mediated regulatory network in the BR signaling pathway. OsbHLH079 enhances brassinosteroid signaling by upregulating genes encoding positive regulators of the BR signaling pathway, such as *OsBZR1*, and *ILI1*, and downregulating *DLT*, which also encodes a positive regulator of BR signaling and downregulated directly by OsBZR1. Then, altered expressions of BR-responsive genes such as *OsEXPA*, and *OsXTH* produce changes in leaf angle and grain length. Arrows and bars indicate positive and negative regulation, respectively. Solid and dashed lines indicate direct regulation and possible feed-forward regulation, respectively.
