Role of Endophytic Bacteria in the Remobilization of Leaf Nitrogen Mediated by CsEGGT in Tea Plants (Camellia sinensis L.).

As an important economic plant, tea (Camellia sinensis) has a good economic value and significant health effects. Theanine is an important nitrogen reservoir, and its synthesis and degradation are considered important for nitrogen storage and remobilization in tea plants. Our previous research indicated that the endophyte CsE7 participates in the synthesis of theanine in tea plants. Here, the tracking test confirmed that CsE7 tended to be exposed to mild light and preferentially colonized mature tea leaves. CsE7 also participated in glutamine, theanine, and glutamic acid circulatory metabolism (Gln-Thea-Glu) and contributed to nitrogen remobilization, mediated by the γ-glutamyl-transpeptidase (CsEGGT) with hydrolase preference. The reisolation and inoculation of endophytes further verified their role in accelerating the remobilization of nitrogen, especially in the reuse of theanine and glutamine. This is the first report about the photoregulated endophytic colonization and the positive effect of endophytes on tea plants mediated and characterized by promoting leaf nitrogen remobilization.

γ-Glutamyl-transpeptidase CsGGT2 functions as light-activated theanine hydrolase in tea plant (Camellia sinensis L.).

Theanine is an important secondary metabolite endowing tea with umami taste and health effects. It is essential to explore the metabolic pathway and regulatory mechanism of theanine to improve tea quality. Here, we demonstrated that the expression patterns of CsGGT2 (γ-glutamyl-transpeptidase), participated in theanine synthesis in vitro in our previous research, are significantly different in the aboveground and underground tissues of tea plants and regulated by light. Light up-regulated the expression of CsHY5, directly binding to the promoter of CsGGT2 and acting as an activator of CsGGT2, with a negative correlation with theanine accumulation. The enzyme activity assays and transient expression in Nicotiana benthamiana showed that CsGGT2, acting as bifunctional protein, synthesize and degrade theanine in vitro and in planta. The results of enzyme kinetics, Surface plasmon resonance (SPR) assays and targeted gene-silencing assays showed that CsGGT2 had a higher substrate affinity of theanine than that of ethylamine, and performed a higher theanine degradation catalytic efficiency. Therefore, light mediates the degradation of theanine in different tissues by regulating the expression of the theanine hydrolase CsGGT2 in tea plants, and these results provide new insights into the degradation of theanine mediated by light in tea plants.

Characterization of CsTSI in the Biosynthesis of Theanine in Tea Plants (Camellia sinensis).

Theanine is a unique major amino acid in tea plants responsible for umami taste and mental health benefits of tea. However, theanine biosynthesis and physiological role in tea plants are not fully understood. Here, we demonstrate that tea plant theanine synthetase is encoded by a glutamine synthetase gene CsTSI. The expression pattern of CsTSI is closely correlated with theanine and glutamine levels in various tissues. CsTSI transcripts were accumulated in root tip epidermal cells, pericycle and procambial cells, where CsTSI presents as a cytosolic protein. Ectopic expression of the gene in Arabidopsis led to greater glutamine and theanine production than controls when fed with ethylamine (EA). RNAi knockdown or overexpression of CsTSI in tea plant hairy roots reduced or enhanced theanine and glutamine contents, respectively, compared with controls. The CsTSI recombinant enzymes used glutamate as an acceptor and ammonium or EA as a donor to synthesize glutamine and theanine, respectively. CsTSI expression in tea roots responded to nitrogen supply and deprivation and was correlated with theanine contents. This study provides fresh insights into the molecular basis for the biosynthesis of theanine, which may facilitate the breeding of high-theanine tea plants for improving the nutritional benefit of tea.

Endophytic Bacteria as Contributors to Theanine Production in Camellia sinensis.

Theanine is the most abundant non-protein amino acid in Camellia sinensis, but it is not known how a tea plant accumulates such high levels of theanine. The endophyte isolated from in vitro grown plantlets of C. sinensis cultivars was identified as Luteibacter spp., showing strong biocatalytic activity for converting both glutamine and ethylamine to theanine. Theanine was secreted outside of the bacteria. The endophyte isolated from in vitro plantlets of Camellia oleifera cultivar was identified as Bacillus safensis and did not convert glutamine and ethylamine to theanine. Enzymatic assays in vitro indicated that γ-glutamyltranspeptidases rCsEGGTs from the endophyte Luteibacter strains converted glutamine and ethylamine to theanine at higher rates than rCsGGTs from C. sinensis. This is the first report on theanine biosynthesis by an endophyte from C. sinensis, which provides a new pathway to explore the mechanism of theanine biosynthesis in C. sinensis and the interactions between an endophyte and tea plants.