The mitochondrial carrier CsTHS1 acts as a gatekeeper of theanine accumulation in late-spring new shoots of tea plants

Theanine is a core secondary metabolite responsible for the sensory qualities and health benefits of tea. Theanine levels are high in new tea plant (Camellia sinensis) shoots that arise during early spring, but then significantly decrease in late spring, causing a rapid decline in the quality of green tea processed from the late-spring harvest. However, the molecular mechanisms underlying this seasonal decrease in theanine levels remain unknown. In a previous genetic screen, we identified the YFR045W yeast mutant that displayed a hypersensitivity to theanine feeding due to enhanced theanine accumulation. YFR045W encodes a putative mitochondrial carrier and was designated Theanine Hypersensitive 1 (THS1). Expression of CsTHS1, the functional homolog of THS1 in tea plants, rescued this yeast mutant phenotype. Importantly, CsTHS1 expression is induced in late-spring new shoots, and CsTHS1 exhibits a high affinity for theanine. Yeast mitochondria expressing CsTHS1 demonstrate increased theanine transport activity, and recombinant CsTHS1 proteo-liposomes can also transport theanine. Additionally, CsTHS1 overexpression or repression in new tea shoots significantly decreased or increased theanine accumulation, respectively. Our findings establish a relationship between CsTHS1-mediated theanine transport into mitochondria and the observed reduction in theanine accumulation in the late-spring new tea shoots. Our study supports a mechanism whereby CsTHS1 mediates theanine entry into mitochondria for degradation by the mitochondria-localized γ-glutamyl transpeptidase (CsGGT2).