Characterization of MaWOX genes in mulberry reveals the role of MaWOX4 in promoting shoot regeneration of leaf explants

• Eleven MaWOX genes were identified in mulberry and phylogenetic analysis revealed their conservation with WOX homologs in other plant species. • Five members of MaWOX family were found related to the phytohormone-induced shoot regeneration of mulberry leaf explants. • The MaWOX4 protein displayed nuclear re-localization in the division of mulberry mesophyll protoplasts, indicating possible function in this process. • Overexpression of MaWOX4 improved the shoot regeneration ability of tobacco and led to phenotypic variation. The WUSCHEL-related homeobox (WOX) transcription factors are essential plant growth and development regulators. However, little is known about WOX genes in mulberry, a perennial woody plant valued for its agricultural and medicinal uses. In this study, we identified a relatively compact and conserved WOX gene family in the mulberry through bioinformatics and tissue-specific gene expression analyses. Among the eleven MaWOX genes characterized in Morus alba ' Heyebai', five ( MaWUS, MaWOX1, MaWOX3, MaWOX4, MaWOX13 ) were highly associated with the phytohormone-induced shoot regeneration of mulberry leaf explants. Transient gene expression assays in mulberry protoplasts confirmed typical nuclear localization for three of these genes, suggesting their role as active transcription factors. Notably, MaWOX4 showed a shift from universal to nuclear localization in protoplasts undergoing cell division, pointing to its involvement in cell cycle regulation. Constitutive overexpression of MaWOX4 in tobacco significantly increased the transformation efficiency and shoot regeneration ability, highlighting its critical role in these processes. These findings provide valuable insights for further studies aiming at uncovering the regulatory mechanisms behind shoot regeneration in woody plants and highlight MaWOX4 as a potential candidate gene for improving genetic transformation, supporting gene functional studies, and advancing the breeding of mulberry.