The regulatory module MdCPCL-MdILR3L mediates the synthesis of ascorbic acid and anthocyanin in apple.

Apple (Malus domestica Borkh.) is one of the most economically valuable fruit crops globally and a key dietary source for various nutrients. However, the levels of ascorbic acid (AsA) and anthocyanin, essential micronutrients for human health, are extremely low in the pulp of commonly cultivated apple varieties. In the present study, the second-generation hybrid strain of Xinjiang red-fleshed apple ('Zihong No. 1' × 'Gala') was used as the test material. The results revealed that AsA content was significantly higher in red-fleshed apple pulp than in non-red-fleshed varieties, and the expression of MdGLDH, a key gene in the D-mannose/L-galactose pathway, correlated strongly with AsA levels. Using the promoter of MdGLDH as bait, an R3-type MYB transcription factor (TF), MdCPC-like, was identified through yeast one-hybrid screening. Further analysis revealed that the overexpression of MdCPCL increased the AsA and anthocyanin levels in both callus and fruits, whereas MdCPCL knockdown led to a reduction in their levels. Moreover, the interaction between MdCPCL and the bHLH TF MdILR3-like was confirmed, forming the MdCPCL-MdILR3L complex. This complex significantly enhanced the transcription of downstream target genes MdGLDH and MdANS, promoting the synthesis of AsA and anthocyanins. This study contributes to further enrich the anabolic pathways of AsA and anthocyanin in apples and provides a theoretical foundation for the quality breeding of red-fleshed apple varieties.