Theanine, a tea plant-specific non-proteinogenic amino acid, is the most abundant free amino acid in tea leaves. It is also one of the most important quality components of tea because it endows the “umami” taste, relaxation-promoting, and many other health benefits of tea infusion. Its content in tea leaves is directly correlated with the quality and price of green tea. Theanine biosynthesis primarily occurs in roots and is transported to new shoots in tea plants. Recently, great advances have been made in theanine metabolism and transport in tea plants. Along with the deciphering of the genomic sequences of tea plants, new genes in theanine metabolic pathway were discovered and functionally characterized. Theanine transporters were identified and were characterized on the affinity for: theanine, substrate specificity, spatiotemporal expression, and the role in theanine root-to-shoot transport. The mechanisms underlying the regulation of theanine accumulation by: cultivars, seasons, nutrients, and environmental factors are also being rapidly uncovered. Transcription factors were identified to be critical regulators of theanine biosynthesis. In this review, we summarize the progresses in theanine: biosynthesis, catabolism, and transport processes. We also discuss the future studies on theanine in tea plants, and application of the knowledge to crops to synthesize theanine to improve the health-promoting quality of non-tea crops.

Theanine, one of the most important components of teas, confers the umami taste and relaxation effect of tea infusion. As a non-proteinogenic amino acid, theanine solely accumulates to high levels in tea plants (Camellia sinensis L.); however, the underlying mechanism remains largely unknown. Theanine is biosynthesized from ethylamine and glutamate by CsTSI (Theanine Synthetase I), with ethylamine being synthesized from alanine, by alanine decarboxylase. Although CsAlaDC exhibited alanine decarboxylase activity, in vitro, the in vivo role in tea plants has not been characterized. In this study, we found ethylamine content in tea roots to be highly correlated with theanine content (r=0.883, p <0.01), and expression levels of CsAlaDC were similarly highly correlated with the accumulation of ethylamine (r=0.903, p <0.01) and theanine (r=0.881, p <0.01). CsAlaDC-expressing tobacco leaves produced high levels of ethylamine, but did not produce detectable theanine. However, high levels of theanine were produced in leaves co-expressing CsAlaDC and CsTSI. These results indicate CsAlaDC and CsTSI act coordinately in determining the high level of theanine biosynthesis. This is also the first report to synthesize theanine in a non-tea plant, which offers the potential to generate relaxation-promoting foods by synthesizing theanine in crops.

Theanine, a unique non-proteinogenic amino acid, is one of the most abundant secondary metabolites in tea. Its content largely determines green tea quality and price. However, its physiological roles in tea plants remain largely unknown. Here, we showed that salt stress significantly increased the accumulation of glutamate, glutamine, alanine, proline, and γ-aminobutyric acid, as well as theanine, in the new shoots of tea plants. We further found that salt stress induced the expression of theanine biosynthetic genes, including CsGOGATs, CsAlaDC, and CsTSI, suggested that salt stress induced theanine biosynthesis. Importantly, applying theanine to the new shoots significantly enhanced the salt stress tolerance. Similar effects were also found in a model plant Arabidopsis. Notably, exogenous theanine application increased the antioxidant activity of the shoots under salt stress, suggested by reduced the reactive oxygen species accumulation and lipid peroxidation, as well as by the increased SOD, CAT, and APX activities and expression of the corresponding genes. Finally, genetic evidence supported that catalase-mediated antioxidant scavenging pathway is required for theanine-induced salt stress tolerance. Taken together, this study suggested that salt stress induces theanine biosynthesize in tea plants to enhance the salt stress tolerance through a CAT-dependent redox homeostasis pathway.

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.