MdCML50 Promotes the Phosphorylation of MdWRKY6 to Mediate Calcium-Inhibited Ethylene Biosynthesis in Postharvest Apple Fruit

Ethylene is the key hormone regulating postharvest ripening and senescence in apple (Malus domestica). Calcium ions (Ca²⁺) treatment inhibits ethylene biosynthesis in apple fruit, thereby prolonging shelf life; however, the molecular mechanisms underlying Ca²⁺ mediated inhibition of ethylene biosynthesis remain largely elusive. In this study, we identified a calmodulin-like protein, MdCML50, which functions as a Ca²⁺-responsive sensor and negatively regulates ethylene production in postharvest apple fruit. CaCl₂ treatment significantly induced MdCML50 expression and suppressed ethylene biosynthesis by downregulating the transcription of the key biosynthetic genes MdACS1 and MdACO1. We further demonstrated that MdWRKY6 acts as a positive regulator of fruit ripening by directly binding to the promoters of MdACS1 and MdACO1 to activate their transcription. MdCML50 physically interacts with MdWRKY6, and this interaction is markedly enhanced by Ca²⁺. Notably, Ca²⁺-activated MdCML50 promotes the phosphorylation of MdWRKY6, leading to its degradation through the 26S proteasome pathway. The reduced abundance of MdWRKY6 weakens its transcriptional activation of MdACS1 and MdACO1, ultimately resulting in suppressed ethylene biosynthesis. Taken together, our results uncover a previously uncharacterized Ca²⁺–MdCML50–MdWRKY6 regulatory module that fine-tunes ethylene production via post-translational control of transcription factor stability. This study provides important insights into Ca²⁺-mediated postharvest fruit preservation and offers a promising biotechnological strategy for improving apple storage life and shelf quality.