ClMYB5-mediated regulation of ClVHP1 expression controls citric acid storage in wampee fruit

Fruit acidity, a crucial determinant of flavor and quality, is primarily governed by the content and composition of organic acids, with citric acid playing a dominant role. To unravel the molecular basis of citric acid accumulation, we analyzed four wampee (Clausena lansium) cultivars-sweet-type 'Huami' and 'Baitang' and sweet-sour-type 'Huami 2' and 'Wuhe'-by integrating transcriptomic data with organic acid profiling. Candidate genes were systematically screened based on their expression patterns and correlations with acid content.

Transcriptome analysis identified three key regulators of citric acid accumulation: ClVHP1 (encoding a vacuolar H⁺-phosphatase), ClMYB5, and ClbHLH42, all exhibiting strong expression-acidity correlations. Subcellular localization confirmed the nuclear presence of ClMYB5 and ClbHLH42, while transient overexpression in tomato validated their functional roles, consistently elevating citric acid levels. Conversely, virus-induced gene silencing (VIGS) of these factors markedly reduced acid accumulation. Mechanistically, we demonstrated that ClMYB5 directly binds the ClVHP1 promoter to activate its expression, thereby enhancing vacuolar acid sequestration via ClVHP1's proton-pump activity. However, the ClbHLH42 participates in the acid regulation but indirect.

Our study establishes a coordinated regulatory module wherein ClMYB5, ClbHLH42, and ClVHP1 collectively govern citric acid storage in wampee. The discovery of ClMYB5-mediated ClVHP1 activation provides novel insights into vacuolar acid partitioning. These findings not only advance the understanding of organic acid metabolism but also offer strategic targets for precision breeding of fruit quality traits.