ClPH4-like coordinately regulates citric acid and anthocyanin accumulation in purple wampee

Abstract Citric acid and anthocyanins are key metabolites that determine fruit flavor and coloration. Although the accumulation mechanisms of citric acid and anthocyanins have been known in citrus, little is known about the cross-regulatory mechanism between the two important metabolites. In this study, we identified a purple wampee (Clausena lansium) that exhibits simultaneous accumulation of citric acid and anthocyanins in fruit. Functional analyses confirmed that the ClPH4 positively regulate citric acid accumulation, however, the homologous gene of Ruby1, the activator of anthocyanin biosynthesis, has lost the capacity to induce anthocyanin accumulation in the purple wampee. We subsequently identified a PH4-Like based on gene family and expression analyses. Stable overexpression of ClPH4-like in citrus, along with transient silencing in purple wampee pulp, demonstrated that it can simultaneously induce both anthocyanin and citric acid accumulation. Moreover, PH4-Like is specifically and highly expressed in purple wampee, consistent with the observation that its promoter activity is much higher in purple wampee than in other citrus species. Biochemical assays showed that PH4-Like binds to the promoters of Flavonoid 3’-Hydroxylase (F3’H), Dihydroflavonol 4-reductase (DFR), Anthocyanidin synthase gene (ANS) and the proton pump gene PH5 and VHA-a2, the key genes in the anthocyanin and citric acid accumulation. Together, our findings uncover a key gene that coordinately regulates citric acid and anthocyanins accumulation, providing insight into the genetic events for the diversification of fruit color and tastes in Aurantioideae, and offering valuable targets for simultaneous improvement of fruit quality.