MdSPL1-MdNF-YB4-MdCKX7 Module Positively Regulates Saline-Alkali Tolerance by Mediating Cytokinin Levels in Apple

Saline-alkali stress is a major abiotic factor that compromises plant growth, crop yield, and quality. Cytokinin plays an essential role in plant responses to saline-alkali stress; however, the underlying molecular mechanisms remain unclear in apple (Malus domestica). In this study, CYTOKININ OXIDASE/DEHYDROGENASE7 (MdCKX7) was identified as a positive regulator of saline-alkali tolerance in apple, encoding a functional CKX enzyme that mediates cytokinin degradation. Consistently, applying exogenous lovastatin, an inhibitor of cytokinin biosynthesis, alleviated the saline-alkali-sensitive phenotype in MdCKX7-silenced plants by promoting stomatal closure through cytokinin degradation. Molecular genetic assays revealed that apple transcription factor MdSPL1 is involved in the response to saline-alkali stress by directly activating the expression of MdCKX7. Overexpression of MdSPL1 enhanced saline-alkali tolerance by modulating photosynthetic efficiency and stomatal aperture, whereas its silencing resulted in increased sensitivity. Furthermore, overexpression of MdCKX7 in the MdSPL1-RNAi background rescued the saline-alkali-sensitive phenotype. Further analysis revealed that MdNF-YB4, a positive regulator of saline-alkali tolerance, interacts with MdSPL1 to facilitate its transcriptional activation of MdCKX7. Collectively, our study reveals the MdSPL1-MdNF-YB4-MdCKX7 regulatory module that orchestrates cytokinin-mediated saline-alkali tolerance in apple, providing valuable molecular tools for the breeding of saline-alkali-tolerance apple varieties.