PalCYCD3;3 breaks axillary bud dormancy and promotes shoot branching through activation by PalBES1 in the BR signaling pathway.

The development of lateral branches in plants is intricately modulated by phytohormonal signaling networks; however, the functional role of D-type cyclins (CYCDs) within this regulatory scheme is not yet fully elucidated. Recent investigations have identified brassinosteroids (BRs) as pivotal regulators of cell cycle dynamics, yet their interactions with CYCD-mediated pathways in meristematic activity require systematic investigation. This research aimed to clarify the function of PalCYCD3; 3 in the regulation of axillary meristem (AM) and its interaction with BR signaling in Populus alba. Histochemical GUS staining demonstrated the specific localization of PalCYCD3;3 within the organizing centers of both the shoot apical meristem (SAM) and AM. PalCYCD3;3-overexpressing P. alba displayed a significant increase in lateral branch formation compared to wild-type counterparts, alongside a marked reduction in AM cell size, mirroring the effects observed in wild-type plants treated with 1 μM brassinolide (BL). Treatment with BL was shown to upregulate both PalCYCD3;3 and the BR signaling mediator PalBES1 in axillary buds. Dual-luciferase reporter assays and electrophoretic mobility shift assays verified the direct interaction of PalBES1 with the promoter of PalCYCD3;3. Additionally, yeast two-hybrid screening combined with bimolecular fluorescence complementation confirmed the physical associations between PalCYCD3; 3 and key cell cycle regulators PalCDKA; 1, PalCDKB1; 1, PalCDKD; 1, and PalCDKE; 1. Our findings establish that BR signaling activates PalCYCD3; 3-CDK complexes through PalBES1-mediated transcriptional regulation, thereby promoting AM cell proliferation and lateral branch development.