The Interaction Between CitMYB52 and CitbHLH2 Negatively Regulates Citrate Accumulation by Activating CitALMT in Citrus Fruit

Citric acid plays significant roles in numerous physiological processes in plants, including carbon metabolism, signal transduction, and tolerance to environmental stress. For fruits, it has a major effect on fruit organoleptic quality by directly influencing consumer taste. Citric acid in citrus is mainly regulated by the balance between synthesis, degradation, and vacuolar storage. The genetic and molecular regulations of citric acid synthesis and degradation have been comprehensively elucidated. However, the transporters for citric acid in fruits are less well understood. Here, an aluminum-activated malate transporter, CitALMT, was characterized. Transient overexpression and stable transformation of CitALMT significantly reduced citrate concentration in citrus fruits and transgenic callus. Correspondingly, transient RNA interference-induced silencing of CitALMT and increased citrate significantly, indicating that CitALMT plays an important role in regulating citrate concentration in citrus fruits. In addition, dual-luciferase assays indicated that CitMYB52 and CitbHLH2 could trans-activate the promoter of CitALMT. EMSA analysis showed that CitbHLH2 could physically interact with the E-box motif in the CitALMT promoter. Bimolecular fluorescence complementation assays, yeast two-hybrid, coimmunoprecipitation and transient overexpression, and RNAi assay indicated that the interaction between CitMYB52 and CitbHLH2 could synergistically trans-activate CitALMT to negatively regulate citrate accumulation.