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 (gamma-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.