The MdHOX32/MdARR20-MdWUS1 module regulates leaf regeneration in apple

The main limitation of molecular breeding in fruit trees is the difficulty in genetic transformation. The regenerative capacity of explants is vital for the success of genetic transformation. Our previous study found that the expression of MdAIL5 was significantly and positively correlated with the rate of adventitious shoot (AS) regeneration in different genotypes of apple ( Malus × domestica ). However, the function and regulatory pathway of MdHOX32 , a downstream gene directly regulated by MdAIL5, remain unclear. Here we showed that the overexpression of MdHOX32 significantly improved the efficiency of AS regeneration in apple leaves. The interaction between MdHOX32 and a type-B Arabidopsis Response Regulator (B-ARR) protein was identified through yeast two-hybrid (Y2H) screening combined with bimolecular fluorescence complementation (BiFC) assays and co-inmunoprecipitation (Co-IP) assays. Further investigations using yeast one-hybrid (Y1H), the electrophoretic mobility shift assay (EMSA), and dual luciferase reporter assay (LUC) demonstrated that MdHOX32 regulated the expression of MdWUS1 by interacting with MdARR20, thereby enhancing the efficiency of leaf AS regeneration. The present study proposed that the MdHOX32/MdARR20–MdWUS1 module regulated the pathway of AS regeneration in apple leaves, thereby providing a theoretically grounded framework for enhancing the efficiency of genetic modification of apple.