Cytokinin drives apple branching through the MdWUS2–MdSPL9–MdTCP regulatory module

Branching is a fundamental determinant of plant architecture and a key agronomic trait. Shoot branching originates from axillary meristems and is tightly regulated by a complex network of genetic, hormonal, and environmental cues. Cytokinin (CTK) promotes apple branching by regulating axillary bud outgrowth. However, the underlying genetic and molecular mechanisms remain unclear. Here, we identified 34 members of the MdSBP transcription factor family through genome-wide analysis. Phylogenetic analysis revealed that MdSBP24 (designated as MdSPL9) is closely related to the known branching regulators AtSPL9/15 in Arabidopsis and OsSBP14 in rice. Gene expression and promoter activity assays showed that MdSPL9 is downregulated by CTK. MdSPL9-RNAi apple plants exhibited increased branching, whereas overexpression of MdSPL9 in the Arabidopsis spl9 mutant reduced branch number and rosette leaf formation, indicating a role in inhibiting branching and axillary meristem formation. MdSPL9 directly activated MdTCP12 and MdTCP18 ( TB1 / BRC1 -like genes), which were also significantly downregulated by CTK. Moreover, MdSPL9 physically interacted through its N-terminal region with the transcriptional repressor MdWUS2, which is activated by CTK and promotes branching. This interaction occurred independently of the C-terminal EAR motif in MdWUS2. Transient assays showed that MdWUS2 suppressed MdSPL9-mediated transcriptional activation of MdTCP12 / 18 in an EAR motif-dependent manner. Collectively, our findings reveal a CTK-regulated transcriptional module in which MdWUS2 antagonizes MdSPL9 activity, thereby modulating the expression of MdTCP12/18 to control apple branching. This study provides novel insights into the hormonal regulation of axillary bud outgrowth and potential molecular targets for optimizing apple tree architecture.