Adventitious shoot (AS) regeneration accelerates plant reproduction and genetic transformation. WOX11 is involved in many biological processes, but its regulation of AS regeneration has not been reported. Here, we showed that the genotype and CK/IAA ratio of apple leaves were the key factors that affected their capacity for AS formation. Moreover, the expression level of MdWOX11 was negatively correlated with the capacity for AS formation. Phenotypic analysis of MdWOX11 transgenic plants showed that overexpression of MdWOX11 inhibited AS formation. Endogenous hormone analysis demonstrated that the contents of auxin (IAA), cytokinin (CK), and abscisic acid (ABA) were higher in MdWOX11-RNAi plants than in MdWOX11-OE transgenic plants. We used RNA sequencing to examine the transcriptional responses of genes in MdWOX11-RNAi and MdWOX11-OE transgenic apple plants at different AS stages. We identified 8066 differentially expressed genes and focused our analysis on those involved in the IAA, CK, ABA, and gibberellin (GA) hormone signaling pathways. The expression of genes related to the CK signaling pathway and shoot development was higher in GL-3 than in MdWOX11-OE transgenic plants during the callus and AS emergence stages. However, the expression of MdCKX5 was higher in MdWOX11-OE transgenic plants than in GL3 and MdWOX11-RNAi transgenic plants. Yeast one-hybrid (Y1H) assays, dual-luciferase reporter assays, and ChIP-qPCR showed that MdWOX11 binds to the promoter of MdCKX5, and a dual-luciferase reporter assay showed that MdWOX11 enhanced the promoter activity of MdCKX5. We concluded that MdCKX5 acts downstream of MdWOX11 to control AS formation, and we built a regulatory model of the suppression of AS formation by MdWOX11 in apple.

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