The plasma membrane-localized CsMAC9F1 mediates citrate accumulation in citrus fruit

Citrate, the predominant organic acid in citrus fruits, is a major determinant of fruit acidity. While significant progress has been made in elucidating the mechanisms of citrate accumulation, the comprehensive molecular regulatory network remains incompletely understood. In this study, 'Jinxiu' Bingtang sweet orange (Citrus sinensis) is a bud mutant derived from 'Qianyang' Bingtang sweet orange and exhibits an extremely low-acidity phenotype. CsMAC9F1 expression was associated with citrate accumulation in both cultivars. Unlike previously characterized tonoplast-localized proteins, CsMAC9F1 localizes to the plasma membrane, suggesting its involvement in a novel pathway regulating citrate accumulation. Functional analyses demonstrated that CsMAC9F1 acts as a positive regulator of citrate accumulation in citrus. We further identified a bHLH transcription factor, CsbHLH113, which binds the CsMAC9F1 promoter and activates its expression. Overexpression and silencing analyses across citrus, kumquat, and heterologous tomato systems consistently supported the role of CsbHLH113 in promoting citrate accumulation through CsMAC9F1. Together, our findings reveal a CsbHLH113-CsMAC9F1 regulatory module that adds a plasma membrane-associated branch to the citrus acidity network and provides new insights into the transcriptional control of fruit acidity.