CsGDH2.1 negatively regulates theanine accumulation in the late-spring tea plants (<i>Camellia sinensis</i> var. <i>sinensis</i>)

Abstract Theanine, a unique and the most abundant non-proteinogenic amino acid in tea plants, endows tea infusion with the umami taste and anti-stress effects. Its content in tea highly correlates with green tea quality. Theanine content in the new shoots of tea plants is high in middle spring and greatly decreases in late spring. However, how the decrease is regulated is largely unknown. In a genetic screening, we observed that a yeast mutant glutamate dehydrolase 2 (gdh2) was hypersensitive to 40 mM theanine and accumulated more theanine. This result implied a role of CsGDH2s in theanine accumulation in tea plants. Therefore, we identified the two homologs of GDH2, CsGDH2.1 and CsGDH2.2, in tea plants. Yeast complementation assay showed that the expression of CsGDH2.1 in yeast gdh2 mutant rescued the theanine hypersensitivity and hyper-accumulation of this mutant. Subcellular localization and tissue-specific expression showed CsGDH2.1 localized in the mitochondria and highly expressed in young tissues. Importantly, CsGDH2.1 expression was low in early spring, and significantly increased in late spring, in the new shoots of tea plants. These results all support that CsGDH2.1 regulates theanine accumulation in the new shoots. Moreover, the in vitro enzyme assay showed that CsGDH2.1 had glutamate catabolic activity, and knock-down CsGDH2.1 expression increased glutamate and theanine accumulation in the new shoots of tea plants. These findings suggested that CsGDH2.1-mediated glutamate catabolism negatively regulates theanine accumulation in the new shoots in late spring, and provided a functional gene for improving late-spring green tea quality.