The SPL8-YAB3/TCP19 module integrates transcriptional and protein-protein regulatory mechanisms to orchestrate anther development in Arabidopsis.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 8 (SPL8) is a plant-specific transcription factor essential for anther development, but its regulatory partners and downstream mechanisms remain largely unknown. We identified 26 SPL8-interacting transcription factors by yeast two-hybrid screening and bimolecular fluorescence complementation (BiFC) assays, with YABBY (YAB) and TEOSINTE BRANCHED 1/CYCLOIDEA/PCF (TCP) family emerging as the predominant interactors. Among these, YAB3 and TCP19 were selected for detailed characterization. Their interactions with SPL8, as well as with each other, were confirmed in plant nuclei by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM). Chromatin immunoprecipitation sequencing (ChIP-seq), ChIP-qPCR, yeast one-hybrid, and dual-luciferase assays demonstrated that SPL8 directly binds the regulatory regions of YAB3 and TCP19 and activates their transcription. Dexamethasone-inducible expression assays further showed that this induction occurs independently of de novo protein synthesis. Expression analyses revealed overlapping spatial and temporal patterns of SPL8, YAB3, and TCP19 during early anther development, particularly in tapetal cells, microspore mother cells, and vascular tissues. Genetic and cellular analyses of promoter-driven SRDX repression lines showed that SPL8, YAB3, and TCP19 regulate anther morphogenesis, tapetal differentiation, meiotic progression, and microspore development. Although individual family members exhibited functional redundancy, their repression caused severe developmental defects and male sterility. Together, these findings establish the SPL8-YAB3/TCP19 module as a multilayered regulatory system in which SPL8 controls anther development through both transcriptional activation of YAB3 and TCP19 and physical interaction with these proteins, expanding the regulatory framework of SPL-mediated reproductive development.