Vacuolar metabolomic and proteomic profiling reveals the vacuole composition of ripe juice sacs and functions of CsTST2 and CsERDL6 in sugar accumulation in citrus

Vacuolar composition, particularly the type and abundance of metabolites and tonoplast proteins, critically determines fruit quality and flavor. However, the specific vacuolar composition of fruits with different flavors at the fully ripe stage, especially regarding sugar accumulation, remains unclear. In this study, we established an optimized protocol to overcome technical barriers in isolating intact vacuoles from fresh ripe citrus juice sacs of four citrus cultivars: NFMJ, Nanfeng tangerine (Citrus reticulata ‘97-2’); WZMG, Wenzhou satsuma (C. unshiu ‘Miyagawa’); NHER, ‘Newhall’ navel orange (C. sinensis ‘Newhall’); MJY, Majiayou pomelo (C. grandis ‘Xipi Majia’). Subsequently, quasi-targeted metabolomics analysis and 4D-label-free proteomics analysis were conducted, identifying 640 metabolites and 1,782 proteins, respectively. Notably, amino acids, flavonoids, lipids, carbohydrates, and organic acids collectively represented 70% of the total vacuolar metabolites. Pummelo MJY vacuoles exhibited the highest sucrose accumulation, whereas tangerine NFMJ showed minimal sucrose content. Proteomic profiling revealed vacuolar proteins participating in protein fate determination, metabolism, vesicle trafficking, solute transport, and energy supply. Comparative analysis demonstrated significantly greater protein abundance variation between MJY and NFMJ than between other varieties. In total, 158 transport proteins, including sugar-related transporters, were identified, and most of them were more abundant in MJY vacuoles. The protein abundance of CsTST2 and CsERDL6 was greater in MJY vacuoles compared to other varieties, and the relative expression patterns of their encoding genes were consistent with sugar accumulation during fruit ripening. Subcellular localization analysis confirmed their tonoplast localization. Importantly, transgenic tomato fruits overexpressing these genes demonstrated both enhanced gene expression and increased sugar content. This study systematically revealed cultivar-specific vacuolar composition and sugar accumulation strategies in ripe citrus fruits and provided key tonoplast proteins responsible for fruit quality improvement.