The cation/H+ exchanger ChCAX3 fine-tunes fruit ripening by modulating cytosolic Ca2+ homeostasis in Cerasus humilis

Cerasus humilis is a calcium-rich fruit crop with high nutritional value, but its synchronized ripening causes concentrated harvest and short shelf life. In this study, ChCAX3, a vacuolar cation/H+ exchanger, was identified as a key regulator of fruit ripening. ChCAX3 showed high expression in fruit and stem tissues and was upregulated by CaCl2 and ethephon treatments. however, repressed during fruit ripening by ChMYB16. Yeast complementation and Arabidopsis thaliana assays validated its Ca2+ transport activity. Overexpression of ChCAX3 in tomato increased fruit Ca2+ content, delayed coloration, reduced softening, and extended the ripening period by approximately 14 days. ChCAX3-mediated vacuolar Ca2+ sequestration may maintain low cytosolic Ca2+ levels, thereby potentially attenuate the expression and activity of a presumptive Ca2+ sensor ChCBL10 and its interacting kinase ChCIPK3 at the vacuolar membrane. This attenuation of the ChCBL10-ChCIPK3 module was accompanied by reduced expression of ethylene biosynthesis genes (ChACS1, ChACO1) and cell wall degradation-related genes (ChPG1, ChPG2), which may collectively delay the ethylene burst and preserving fruit firmness. Conversely, repression of ChCAX3 by ChMYB16 during late ripening may elevate cytosolic Ca2+, potentially activate the ChCBL10-ChCIPK3 signaling cascade, and accelerated senescence. Overall, these findings demonstrated that ChCAX3 positively regulates Ca2+ accumulation and delays fruit ripening via Ca2+ signaling-mediated modulation of ethylene pathways, providing new insights into Ca2+-hormone crosstalk in fruit physiology and identifying a potential target for extending shelf life and enhancing nutritional quality.