*2.1. OsbHLH079 Increases Leaf Angle in Rice*

To identify new components that regulate plant architecture, we screened a collection of T-DNA insertion lines in rice in the Rice Functional Genomic Express Database [51]. We isolated a new mutant with increased leaf angle phenotype (Figure 1a), and found that an enhancer-trap line, PFG\_3A-01275, which is derived from the Korean *japonica* rice cultivar 'Dongjin (hereafter wild type, WT)' harbors a T-DNA containing four tandem repeats of the CaMV 35S promoter in the promoter of *OsbHLH079* (LOC\_Os02g47660) (Figure 1b). To check whether the T-DNA insertion alters the expression of *OsbHLH079*, we compared *OsbHLH079* transcript levels in various organs between WT and the enhancer-trap T-DNA insertion line. RT-qPCR analysis revealed that the transcript levels of *OsbHLH079* in the T-DNA line were much higher in the leaf blade, leaf sheath, and root, compared with WT, although the degrees of overexpression varied among tissues (Figure 1c). Thus, the gain-of-function mutant was termed *osbhlh079-D*.

Next, to characterize the leaf angle phenotype of *osbhlh079-D* in more detail, we compared the leaf angles of the top four leaves between WT and *osbhlh079-D* in field-grown plants at heading stage. The leaf angles of all four top leaves in *osbhlh079-D* were significantly enlarged compared to those in WT, especially those of flag leaves (Figure 1d,e). These results indicate that the overexpression of *OsbHLH079* is closely associated with increase in leaf angle in rice.

To verify if the overexpression of *OsbHLH079* leads to an increase in leaf angle, we generated two independent transgenic rice lines overexpressing the full-length coding sequence of *OsbHLH079* (*35S::OsbHLH079 #2* and *#12*) as well as two individual RNAi-mediated knockdown lines of *OsbHLH079* (*35S::RNAi-OsbHLH079 #4* and *#5*). First, we checked whether the expression of *OsbHLH079* is altered in the *35S::OsbHLH079* and *35S::RNAi-OsbHLH079* lines. RT-qPCR analysis revealed that the transcript levels of *OsbHLH079* were upregulated in two *35S::OsbHLH079* lines (Figure 2a) and downregulated in two *35S::RNAi-OsbHLH079* lines (Figure 2b). Next, we compared the leaf angles of top four leaves among WT, *35S::OsbHLH079*, and *35S::RNAi-OsbHLH079* at heading stage grown under NLD conditions in the paddy field. Indeed, all the leaf angles of *35S::OsbHLH079* were much larger than WT, especially for the flag leaf, as is the case for *osbhlh079-D*. By contrast, all the leaf angles of *35S::RNAi-OsbHLH079* were significantly smaller, except for the flag leaf angle (Figure 2c,d). Collectively, these results suggested that OsbHLH079 increases leaf angle during leaf blade growth.

**Figure 1.** Phenotypic characterization of the *osbhlh079-D* mutant in rice. (**a**) Phenotypes of wild-type (WT) and *osbhlh079-D* at heading stage in plants grown under natural long day (NLD) conditions in the paddy field. Scale bar = 10 cm. (**b**) Schematic diagram illustrating the position of the T-DNA insertion in *OsbHLH079* (LOC\_Os02g47660). Open boxes and filled boxes represent the untranslated region and coding sequence of *OsbHLH079*, respectively. (**c**) Comparison of the *OsbHLH079* transcript levels between 3-week-old plants of WT and *osbhlh079-D* grown under natural sunlight in the greenhouse. The transcript level of *OsbHLH079* was measured by RT-qPCR and normalized to *UBQ5*. Means and standard deviations were obtained from five biological replicates. (**d**) The leaf angle phenotypes of WT and *osbhlh079-D* at heading stage grown under NLD conditions in the paddy field. Scale bar = 1 cm. (**e**) Statistical analysis of leaf angles between WT and *osbhlh079-D* at heading stage grown under NLD conditions in the paddy field. Means and standard deviations were obtained from ten biological replicates. Significant differences between means were analyzed using Student's *t*-test (\*\*\* *p* < 0.001). These experiments were repeated twice with similar results.

**Figure 2.** The leaf angles of WT, *35S::OsbHLH079*, and *35S::RNAi-OsbHLH079*. (**a**) Relative transcript levels of *OsbHLH079* in WT, *35S::OsbHLH079 #2*, and *35S::OsbHLH079 #12*. (**b**) Relative transcript levels of *OsbHLH079* in WT, *35S::RNAi-OsbHLH079 #4*, and *35S::RNAi-OsbHLH079 #5*. (**a**,**b**) Total RNA was extracted from the 2-cm lamina joint tissues between the leaf blade and leaf sheath of WT, *35S::OsbHLH079 #2*, *35S::OsbHLH079 #12*, *35S::RNAi-OsbHLH079 #4*, and *35S::RNAi-OsbHLH079 #5* at heading stage in plants grown under NLD conditions in the paddy field. Relative expression levels of *OsbHLH079* were determined by RT-qPCR analysis and normalized to *UBQ5*. Means and standard deviations were obtained from five biological replicates. Differences between means were compared using Student's *t*-test (\*\* *p* < 0.01). (**c**) Plant phenotypes of WT, *35S::OsbHLH079 #2*, *35S::OsbHLH079 #12*, *35S::RNAi-OsbHLH079 #4*, and *35S::RNAi-OsbHLH079 #5* at heading stage in plants grown under NLD conditions in the paddy field. Scale bar = 10 cm. (**d**) Statistical analysis of leaf angles among WT, *35S::OsbHLH079 #2*, *35S::OsbHLH079 #12*, *35S::RNAi-OsbHLH079 #4*, and *35S::RNAi-OsbHLH079 #5* at heading stage in plants grown under NLD conditions in the paddy field. Means and standard deviations were obtained from ten biological replicates. Significant differences between means were analyzed using Student's *t*-test (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001). These experiments were repeated twice with similar results.
