CsVTT1, a PQ-loop vacuolar amino acid transporter, mediates vacuolar theanine storage in tea plant roots under nitrogen supply

Theanine, a unique and major nitrogen storage amino acid in tea plants, is responsible for the tea umami taste and confers numerous health benefits. Despite its importance, the molecular and cellular mechanisms regulating its biosynthesis and accumulation remain poorly understood. In this study, we identified a PQ-loop amino acid transporter, CsVTT1 (Vacuolar Theanine Transporter 1), through integrative analyses including yeast mutant library screening, along with correlation analysis between gene expression and theanine accumulation, and homologous gene complementation. Our findings revealed that CsVTT1 expression is nitrogen-inducible and strongly correlates with theanine accumulation in tea plant roots under different nitrogen regimes. Functional characterization in both yeast and Xenopus laevis oocytes demonstrated that CsVTT1 facilitates cytoplasmic to vacuolar transport of theanine, glutamine, arginine and other amino acids. Genetic manipulation experiments showed that CsVTT1 overexpression significantly enhanced theanine accumulation in tea plant roots, whereas its knockdown reduced theanine levels. Notably, CsVTT1 overexpression in rice greatly increased arginine accumulation, the main nitrogen storage amino acid in non-tea plants, suggesting a conserved role for CsVTT1 in nitrogen metabolism and storage, consistent with its newly identified function in mediating theanine storage in the vacuole. Consequently, CsVTT1 emerges as a promising target for genetic strategies aimed at simultaneously enhancing tea quality and nitrogen use efficiency.