Expression Analysis of Citrate Metabolism-Related Genes Reveals New Insights into High Citrate Accumulation in a Bingtang Orange Bud Mutant (<i>Citrus sinensis</i> cv. Jinyan)

Understanding the molecular regulation of citric acid accumulation in citrus fruits is crucial, as acidity directly influences fruit flavor, consumer preference, and commercial value. Citric acid is the predominant organic acid in citrus, and its levels are shaped by several factors, including genetic and developmental factors. ‘Jinyan’ Bingtang orange (<i>Citrus sinensis</i> cv. Jinyan) is a novel mutant derived from ‘Jinhong’ Bingtang orange (<i>C. sinensis</i> cv. Jinhong) that has a noticeably sour taste. However, the molecular basis of the increased citrate content in ‘Jinyan’ fruits remains unclear. This study compared the organic acid profiles and expression of citric acid metabolism-related genes between ‘Jinyan’ and ‘Jinhong’ fruit juice sacs throughout fruit development. The trend of citric acid content in both cultivars was similar; however, ‘Jinyan’ consistently presented significantly higher levels than ‘Jinhong’ did from 95 to 215 days after flowering (DAF). After 155 DAF, the transcript levels of citrate biosynthesis-related genes (<i>PEPC1</i>, <i>PEPC2</i>, <i>PEPC3</i>, <i>CS1</i>, and <i>CS2</i>) and citrate transport-related genes (<i>V₁-E1</i>, <i>V₁-E2</i>, <i>V₀-a2</i>, <i>V₀-d</i>, <i>VHP1</i>, <i>VHP2</i>, and <i>CsPH8</i>) were significantly greater in ‘Jinyan’ than in ‘Jinhong’. In contrast, citrate degradation-related genes (<i>NAD-IDH2</i> and <i>NAD-IDH3)</i> were expressed at lower levels than in ‘Jinhong’. Notably, the expression patterns of <i>V₁-E2</i> and <i>CsPH8</i> closely matched the changes in citrate content in both cultivars. These results indicate that, compared with ‘Jinhong’, high citric acid accumulation in the juice sacs of ‘Jinyan’ fruit is likely due to increased citrate synthesis (via upregulated <i>PEPC</i>s and <i>CS</i>s) and increased vacuolar citrate sequestration (via upregulated proton pumps and transporters), coupled with reduced citrate degradation (lower <i>NAD-IDH2/3</i>).