CsCPC, an R3-MYB transcription factor, acts as a negative regulator of citric acid accumulation in Citrus

The citric acid accumulation during fruit ripening determines the quality of fleshy fruits. However, the molecular mechanism underlying citric acid accumulation is not clearly understood yet in citrus due to the scarcity of paired germplasm that exhibits significant difference in organic acid accumulation. Two citrus triploid hybrids with distinct citric acid content in their mature fruits were herein identified from a previously conducted interploidy cross in our group, providing an ideal paired material for studying acid accumulation in citrus. Through a comparative transcriptome analysis of the pulps of the above two triploid hybrids, an R3-MYB transcription factor, CAPRICE (CsCPC), was identified to be a regulator of citric acid accumulation in citrus fruits. Through transgenic experiments involving overexpression (in callus and kumquat fruits) and RNAi (in lemon leaves), we demonstrated that CsCPC suppresses citric acid accumulation by negatively regulating the expression of CsPH1 and CsPH5. Moreover, CsCPC competed with an R2R3-MYB CsPH4 for binding to ANTHOCYANIN1 (CsAN1) and thus disturbed the activation of CsPH1 and CsPH5 that encode vacuolar P-ATPase, which eventually led to a decrease in citric acid content. CsPH4 activated the expression of CsCPC and thus formed an activator-repressor feedback loop, which ultimately inhibited citric acid accumulation in citrus fruit. In summary, this study reveals a new regulatory mechanism of CsCPC-mediated inhibition of citric acid accumulation in citrus fruits, which would support the improvement of citrus fruit quality.