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 (pOsbHLH35::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.

中文翻译：

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OsbHLH35 的严格控制表达对水稻花药发育至关重要

花药发育是一个复杂的过程，由属于不同蛋白质家族的无数转录因子调节。在这项研究中，我们专注于 OsbHLH35 的功能表征，OsbHLH35 是一种调节水稻花药发育的基本螺旋-环-螺旋 (bHLH) TF。过表达 OsbHLH35 的植物呈现小而弯曲的花药，导致种子产量减少 72%。在 OsbHLH35 启动子 (pOsbHLH35::GUS) 控制下表达 GUS 报告基因的水稻转基因植物表明，该 TF 在减数分裂阶段的花药和其他小穗组织中特异性积累。酵母单杂交筛选确定了生长调节因子 (GRF) 家族的三个成员 OsGRF3、OsGRF4 和 OsGRF11，作为 OsbHLH35 的转录调节因子。反式激活试验表明 OsGRF11 负调节拟南芥原生质体中 OsbHLH35 的表达。通过对过表达 OsGRF11 (OsGRF11OE) 的转基因植物的分析，在植物中也观察到了这种调节，证实 OsGRF11 是水稻中 OsbHLH35 的负调节因子。我们的数据表明 OsbHLH35 在水稻的花药发育中起着至关重要的作用，其表达的精细控制对于确保适当的种子生产至关重要。