Tightly controlled expression of OsbHLH35 is critical for anther development in Rice

Abstract Anther development is a complex process regulated by a myriad of transcription factors belonging to distinct protein families. In this study, we focus on the functional characterization of OsbHLH35, a basic Helix-Loop-Helix (bHLH) TF that regulates anther development in rice. Plants overexpressing OsbHLH35 presented small and curved anthers, leading to a reduction of 72% on seed production. Rice transgenic plants expressing GUS reporter gene under the control of OsbHLH35 promoter (OsbHLH35PROM::GUS) showed that this TF specifically accumulates in anthers at the meiosis stage and in other spikelet tissues. Yeast one-hybrid screening identified three members of the Growth-Regulating Factor (GRF) family, OsGRF3, OsGRF4, and OsGRF11, as transcriptional regulators of OsbHLH35. Transactivation assay showed that OsGRF11 negatively regulates OsbHLH35 expression in Arabidopsis protoplasts. This regulation was also observed in planta through the analysis of transgenic plants overexpressing OsGRF11 (OsGRF11OE), confirming that OsGRF11 is a negative regulator of OsbHLH35 in rice. Our data suggest that OsbHLH35 plays an essential role in anther development in rice and the fine control of its expression is crucial to ensure proper seed production.