Two vacuolar proton pumps, PpVHA-c1 and PpVHA-c3, contribute to malate accumulation in peach fruit

Malate is a key determinant of peach ( Prunus persica L.) fruit acidity and quality. Vacuolar H⁺-ATPase (VHA) contributes to malate accumulation in plants, but its function in peach fruit remains unclear. In this study, we identified 23 PpVHA genes in the ‘Zhongyoutao 14’ (CN14) peach genome and manually adjusted three gene models using transcriptome data. Sequence alignment and phylogenetic analysis revealed that each VHA subunit is encoded by at least one gene in peach. Based on malate content, PpVHA-c1 and PpVHA-c3 were selected as candidate genes potentially involved in malate accumulation. Transient transgenic assays revealed that both PpVHA-c1 and PpVHA-c3 promote malate accumulation in peach fruit and Nicotiana benthamiana leaves. Furthermore, we predicted nine and fourteen transcription factors as potential positive regulators of PpVHA-c1 and PpVHA-c3 , respectively. These findings demonstrate that PpVHA-c1 and PpVHA-c3 function as positive regulators of malate accumulation, providing a foundation for dissecting the regulatory network controlling VHA-mediated organic acid metabolism in peach fruit.