Effects of phosphorylation on CsTT12 transport function: A comparative phosphoproteomic analysis of flavonoid biosynthesis in tea plants (<i>Camellia sinensis</i>)

Monomeric flavan-3-ols and their oligomeric forms, proanthocyanidins (PAs), are closely related to the bitterness of tea beverages. Monomeric flavan-3-ols are characteristic flavor compounds in tea. Increasing the content of PAs and anthocyanins enhances the resistance of tea plants to pathogen invasion but decreases the quality of tea beverages. MATE family transporters play a critical role in transferring monomeric flavan-3-ols and anthocyanins into vacuoles for storage or subsequent condensation into PAs. Their activities modulate the ratio of monomeric flavan-3-ols to PAs and increase anthocyanin content in tea plants. In this study, it was observed that the gene expression and protein phosphorylation level of the MATE transporter CsTT12, a vacuole-localized flavonoid transporter, were notably upregulated following exogenous sucrose treatment, promoting PA synthesis in tea plants. Further analysis revealed that overexpression of CsTT12 and CsTT12^S17D significantly increased the content of anthocyanins and PAs in plants, whereas CsTT12^S17A did not. In CsTT12 knockdown plants, PA's accumulation decreased significantly, while monomeric catechin content increased. Moreover, phosphorylation modification enhanced the vacuolar membrane localization of CsTT12, whereas dephosphorylation weakened its vacuolar membrane localization. This study uncovers the crucial role of phosphorylation in flavonoid biosynthesis and provides insights into balancing quality improvements and resistance enhancement.