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.

Citrate Accumulation-Related Gene Expression and/or Enzyme Activity Analysis Combined With Metabolomics Provide a Novel Insight for an Orange Mutant.

'Hong Anliu' (HAL, Citrus sinensis cv. Hong Anliu) is a bud mutant of 'Anliu' (AL), characterized by a comprehensive metabolite alteration, such as lower accumulation of citrate, high accumulation of lycopene and soluble sugars in fruit juice sacs. Due to carboxylic acid metabolism connects other metabolite biosynthesis and/or catabolism networks, we therefore focused analyzing citrate accumulation-related gene expression profiles and/or enzyme activities, along with metabolic fingerprinting between 'HAL' and 'AL'. Compared with 'AL', the transcript levels of citrate biosynthesis- and utilization-related genes and/or the activities of their respective enzymes such as citrate synthase, cytosol aconitase and ATP-citrate lyase were significantly higher in 'HAL'. Nevertheless, the mitochondrial aconitase activity, the gene transcript levels of proton pumps, including vacuolar H(+)-ATPase, vacuolar H(+)-PPase, and the juice sac-predominant p-type proton pump gene (CsPH8) were significantly lower in 'HAL'. These results implied that 'HAL' has higher abilities for citrate biosynthesis and utilization, but lower ability for the citrate uptake into vacuole compared with 'AL'. Combined with the metabolites-analyzing results, a model was then established and suggested that the reduction in proton pump activity is the key factor for the low citrate accumulation and the comprehensive metabolite alterations as well in 'HAL'.