Cytokinin-regulated WUSCHEL promotes lateral bud and vascular cambium development in Pinus yunnanensis

Lateral bud outgrowth and secondary vascular development are regulated by the division and differentiation of meristematic cells. However, the regulatory mechanisms underlying lateral bud outgrowth and meristem activity remain unclear in Pinus yunnanensis. Here, we investigated the processes of lateral bud outgrowth in P. yunnanensis seedlings by analyzing their phenotypic, physiological, and molecular characteristics. Our results provide evidence that lateral bud outgrowth depends on local cytokinin (CK) activity. CK accumulation induced the expression of WUSCHEL (WUS), thereby upregulating the expression of genes related to the cell cycle and proliferation. To further investigate the roles of WUS in this pathway, subsequent studies on PyWUS overexpressing in Arabidopsis significantly accelerated vegetative growth and enhanced shoot branching. In poplar, PyWUS increased the number of embryonic leaves within lateral buds, while defoliation and decapitation triggered earlier and faster bud outgrowth. Notably, PyWUS promoted cambial cells' activity and xylem development in poplar. In addition, PyWUS enhanced root elongation growth in both Arabidopsis and poplar. Yeast two-hybrid assays revealed that PyWUS interacted with PyWOX13, PyWOXA, and ARABIDOPSIS HOMEOBOX8/15 (ATHB8/15), supporting the conserved regulatory network of WUS in meristem development. Our study elucidates the mechanisms underlying lateral bud development in Pinus species. Furthermore, this study demonstrates the conserved function of WUS in regulating lateral bud outgrowth and reveals its novel role in promoting secondary vascular development, providing new insights into its functions in conifers.