*2.3. OsbHLH079 is a Transcription Factor of the Basic Helix-Loop-Helix (bHLH) Family in Rice*

The domains of OsbHLH079 were analyzed using the NCBI-BLASTP program [52]. OsbHLH079 has a conserved basic helix–loop–helix (bHLH) domain from the 174th to 221th amino acids (Figure 4a). Moreover, the bHLH domain was found to be a putative G-box binding type, which directly binds to the G-box motif in the rice genome, in a previous genome-wide analysis [45]. These data suggested that OsbHLH079 is a bHLH-type G-box binding transcription factor. To determine if OsbHLH079 acts as a transcription factor, we first examined its subcellular localization in onion epidermal cells. The *35S::YFP* (control) and *35S::YFP-OsbHLH079* constructs were introduced into the onion epidermal cells by particle bombardment, and, at 18 h after particle bombardment, onion nuclei were stained with DAPI to detect the nucleus. Confocal laser scanning microscopy showed that YFP-OsbHLH079 fusion proteins exclusively localized in the DAPI-stained nuclei, while YFP proteins were detected

throughout the cells (Figure 4b). Next, we performed a transactivation activity assay for OsbHLH079 in yeast. The full-length cDNA of *OsbHLH079* was fused with the yeast GAL4 activation domain in the pGADT7 vector, or with the yeast GAL4 DNA-binding domain in the pGBKT7 vector. Then, the yeast strain AH109, harboring the *HIS3*, *ADE2*, and *LacZ* reporter genes, was co-transformed with a pair of plasmids and plated on each selective medium, as shown in Figure 4c. Only the yeast expressing GAL4BD-OsbHLH079 grew on the selective medium lacking histidine and adenine (Figure 4c). Furthermore, in the β-galactosidase liquid assay, LacZ activity was highly upregulated in the yeast expressing GAL4BD-OsbHLH079 compared to that in the negative control (Figure 4d), indicating that OsbHLH079 has transactivation activity. Taking these observations together, it can be concluded that OsbHLH079 functions as a transcription factor of the basic helix–loop–helix (bHLH) family in rice.

**Figure 4.** OsbHLH079 as a putative transcription factor. (**a**) Domain analysis of the 361-amino-acid-long OsbHLH079 protein. The green box indicates a basic helix–loop–helix domain. (**b**) Subcellular localization of OsbHLH079 in onion epidermal cells. The *35S::YFP* and *35S::YFP-OsbHLH079* constructs were introduced into onion epidermal cells and the cells were analyzed by confocal laser scanning microscopy at 18 h after particle bombardment. Onion nuclei were stained with DAPI. Arrows indicate the nucleus. Scale bar = 50 μm. DAPI, 4- ,6-diamidino-2-phenylindole. (**c**,**d**) Transactivation activity assay of OsbHLH079. The full-length cDNA of *OsbHLH079* was fused with the yeast GAL4 activation domain in the pGADT7 vector, or with the yeast GAL4 DNA-binding domain in the pGBKT7 vector, and the fusion proteins were expressed in the yeast strain AH109. (**c**) Transformed yeasts were grown on the Leu– Trp–, Leu– Trp– His–, and Leu– Trp– His– Ade– agar media for yeast cell survival assay. (**d**) LacZ activity was obtained using the β-galactosidase liquid assay. The relative β-galactosidase activity was obtained by normalizing to the activity level of the negative control. Means and standard deviations were obtained from three biological samples. Significant differences between means were analyzed using Student's *t*-test (\* *p* < 0.05). These experiments were repeated twice with similar results. -, empty vector.
