Effects of exogenous calcium and calcium inhibitors on ascorbic acid biosynthesis and recycling during apple ripening

Ascorbic acid (AsA) is a crucial antioxidant in apple fruit. The L-galactose pathway is considered the primary route for its biosynthesis, whereas the ascorbate-glutathione (AsA-GSH) cycle is the main route for its recycling. Calcium signaling is essential for fruit ripening. To date, relationships between calcium signaling and AsA metabolism in ripening apple fruit are still undetermined. In this work, postharvest apples were treated with calcium chloride (CaCl₂), chlorpromazine (CPZ) and ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and ripened at 20 °C. The results indicated that, compared with the controls, CaCl₂ treatment maintained flesh firmness and reduced respiration rate and ethylene production during ripening. It reduced contents of hydrogen peroxide (H₂O₂) and dehydroascorbic acid (DHA) but increased content of AsA and AsA/DHA ratio during ripening. This treatment additionally promoted activities of L-galactose dehydrogenase (GalDH), L-galactono-1,4-lactone dehydrogenase (GalLDH), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR). Conversely, EGTA and CPZ treatments displayed contrasting results. A total of 71 genes involved in L-galactose and AsA recycling pathways were identified from the apple genome, with 44 genes expressed specifically in fruit tissue. Quantitative real-time PCR (qPCR) analysis revealed that the expression of these genes responded differentially to these three treatments, except for one L-galactose-1-phosphate phosphatase gene (MdGPP4) and MdMDHAR2 whose expression was unaffected by all treatments. These data indicated that calcium signaling play a role in mediating AsA metabolism in ripening apple fruit.