*2.4. OsbHLH079 Enlarges Cell Size in the Adaxial Side of Leaf Lamina Joints by Upregulating Cell Expansion-Related Genes*

In general, the tissue-specific expression of genes is closely associated with their biological functions. Therefore, we first checked the spatial expression patterns of *OsbHLH079* in field-grown (NLD conditions) WT at heading stage. This revealed that *OsbHLH079* is mainly expressed in the stem, node, internode, and lamina joint (Figure 5a). Previous studies showed that several genes controlling leaf angle are highly expressed in the lamina joint, as is the case of *OsbHLH079*, and the degree of leaf inclination is mainly regulated by cell proliferation and/or cell expansion in the lamina joint, especially in the adaxial side of the lamina joint [5,20,21,41,53–57]. Therefore, we speculated that the expression levels of cell proliferation- or expansion-related genes in lamina joint would be altered in *osbhlh079-D*, and thus compared the transcript levels of those genes in the lamina joint between WT and *osbhlh079-D* by RT-qPCR analysis. The expression levels of cell proliferation-related genes, including *OsCDC6*, *OsMCM3*, *OsE2F1*, and *OsCYCA3;1* [58], in the lamina joint were not significantly different between WT and *osbhlh079-D* (Figure 5b). However, the transcript levels of cell expansion-related genes, such as *OsEXPAs* and *OsXTHs* [59,60], were highly upregulated in the lamina joint of *osbhlh079-D* compared to WT (Figure 5c). Therefore, we hypothesized that the increased leaf angle of *osbhlh079-D* might be caused by expansion of cell size, mainly in the adaxial side of lamina joints.

**Figure 5.** Expression of cell cycle- and cell elongation-related genes in *osbhlh079-D*. (**a**) Spatial expression patterns of *OsbHLH079* in WT at the heading stage grown under NLD conditions in the paddy field. The transcript level of *OsbHLH079* was determined by RT-qPCR analysis and normalized to *UBQ5*. Means and standard deviations were obtained from three biological replicates. (**b**) Expression patterns of cell cycle-related genes in the *osbhlh079-D* mutant compared to those in WT. (**b**) Altered expressions of cell elongation-related genes in the *osbhlh079-D* mutant compared to those in WT. (**b**,**c**) Total RNA was extracted from the 2-cm lamina joint segments between leaf blade and leaf sheath of 2-week-old WT and *osbhlh079-D* grown under long day (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 that of *UBQ5*. Means and standard deviations were obtained from three 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. NS, not significant.

Table 079. *D*, we observed longitudinal sections of flag–leaf lamina joints in WT and *osbhlh079-D* by scanning electron microscopy. The cell length on the adaxial side in *osbhlh079-D* was much larger

than WT along the adaxial–abaxial and proximal–distal axes; the abaxial cell size in *osbhlh079-D* was also slightly increased in both axes (Figure 6a–h). These results suggested that OsbHLH079 increases leaf angle by expanding the cell size on the adaxial side of the lamina joint through the upregulation of *OsEXPA* and *OsXTH* genes (Figure 5c).

**Figure 6.** Scanning electron microscopy of the lamina joints of leaves in WT and *osbhlh079-D*. (**a**–**f**) Longitudinal sections of the lamina joint of flag leaf in WT (**a**–**c**) or *osbhlh079-D* (**d**–**f**) at heading stage grown under NLD conditions in the paddy field. (**a**,**d**) Close-up of abaxial regions denoted by rectangles (left side) in (**b**,**e**), respectively. (**c**,**f**) Close-up of adaxial regions denoted by rectangles (right side) in (**b**,**e**), respectively. Scale bar = 200 μm in (**b**,**e**). Scale bar = 50 μm in (**a**,**c**,**d**,**f**). (**g**) Statistical analysis of cell lengths in (**a**,**d**). (**h**) Statistical analysis of cell lengths in (**c**,**f**). (**g**,**h**) Cell lengths along the adaxial–abaxial axis and proximal–distal axis were measured on the abaxial and adaxial sides of lamina joints. Means and standard deviations were obtained from thirty cells. Significant differences between means were analyzed using Student's *t*-test (\* *p* < 0.05, \*\*\* *p* < 0.001). These experiments were repeated twice with similar results.
