Abstract
Early anther morphogenesis is a crucial process for male fertility in plants, governed by the transcription factor SPL. While the involvement of AGAMOUS (AG) in SPL activation and microsporogenesis initiation is well established, our understanding of the mechanisms governing the spatial distribution and precise expression of SPL during anther cell fate determination remains limited. Here, we present novel findings on the abnormal phenotypes of two previously unreported SPL mutants, spl-4 and spl-5, during anther morphogenesis. Through comprehensive analysis, we identified ARF3 as a key upstream regulator of SPL. Our cytological experiments demonstrated that ARF3 plays a critical role in restricting SPL expression specifically in microsporocytes. Moreover, we revealed that ARF3 directly binds to two specific auxin response elements on the SPL promoter, effectively suppressing AG-mediated activation of SPL. Notably, the arf3 loss-of-function mutant exhibits phenotypic similarities to the SPL overexpression mutant (spl-5), characterized by defective adaxial anther lobes. Transcriptomic analysis revealed differential expression of the genes involved in the morphogenesis pathway in both arf3 and spl mutants, with ARF3 and SPL exhibited opposing regulatory effects on this pathway. Taken together, our study unveils the precise role of ARF3 in restricting the spatial expression and preventing aberrant SPL levels during early anther morphogenesis, thereby ensuring the fidelity of the critical developmental process in plants.