Auxin and Cytokinins-Mediated Regulation of Adventitious Shoot Regeneration via the RpARR12/RpARF19-RpWOX4-RpLBD4 Pathway in Robinia pseudoacacia.

Woody plants are fast-growing and ecologically important, with widespread applications in urban landscaping and ecological restoration. However, limited understanding of the molecular mechanisms underlying adventitious shoot regeneration has hindered advances in tissue culture and genetic improvement of woody species. Here, we present a comprehensive framework for elucidating adventitious shoot regeneration in Robinia pseudoacacia. Histological analyses delineated key developmental stages, while RNA-sequencing and hormone profiling highlighted the critical roles of auxin and cytokinins. WGCNA analysis identified hormone-responsive regulatory modules, and chemical inhibition assays confirmed their essential functions in adventitious shoot formation. Notably, we discovered that RpARF19 and RpARR12 directly bind to the RpWOX4 promoter to activate its expression and that RpWOX4, in turn, binds to the RpLBD4 promoter to drive expression. In addition, RpARF19 can directly bind to the promoter of RpLBD4 to activate its expression. Overexpression of RpWOX4 or RpLBD4 significantly enhanced adventitious shoot regeneration. Collectively, these findings establish RpWOX4 as a central integrator of auxin and cytokinins signalling and reveal a novel RpARF19/RpARR12-RpWOX4-RpLBD4 regulatory module that orchestrates adventitious shoot regeneration. This study provides new mechanistic insights into adventitious shoot regeneration in woody species and promising strategies for improving tissue culture and breeding approaches.