Genome-wide identification and transcript analysis of vacuolar-ATPase genes in citrus reveal their possible involvement in citrate accumulation.

The vacuolar H+-ATPase (V-ATPase) proton pump plays an important role in the acidification of vacuoles; however, genes encoding V-ATPase in the citrus genome and their roles in citric acid accumulation remain unclear in citrus fruit. In this study, we found at least one gene encoding subunit A, B, C, D, G, c'', d or e; two genes encoding the subunit E, F, H or a; and four genes encoding subunit c in the citrus genome. Spatial expression analysis showed that most genes were predominantly expressed in the mature leaves and/or flowers but were less expressed in root and juice cells. Two sweet orange (Citrus sinensis) cultivars, 'Anliu' (AL) and 'Hong Anliu' (HAL), which differ in terms of fruit acidity, were used in this study. The citric acid content was significantly higher in 'AL' fruits than in 'HAL' fruits over the entire experimental period (82 days-236 days after full blossom, DAFB). Transcript analysis showed that the transcript levels of most subunit genes, including V1-A, V1-B, V1-C, V1-E1, V1-G, V1-H2 and V0-a2, V0-c", V0-c4, and V0-d, were significantly lower in 'HAL' than in 'AL' fruits during fruit development and ripening. Moreover, ABA injection significantly increased the citric acid content, simultaneously accompanied by the obvious induction of V1-A, V1-C, V1-E1, V1-F1, V1-H2, V0-a1, V0-a2, V0-c1, V0-c2, V0-c4, and V0-d transcription levels. In conclusion, the results demonstrated that V1-A, V1-C, V1-E1, V1-H2, V0-a2, V0-c4, and V0-d may play more roles than other subunit genes in the vacuole acidification of citrus fruits. The lower activity of V-ATPase caused by the transcript reduction of some subunit genes may be one reason for the low citrate accumulation in 'HAL' juice sacs.