Glutamate promotes cold tolerance through CsGLR3.2- and Ca2+-associated CBF signaling in tea plants

Low temperature limits the productivity and quality of tea plants, particularly when spring cold spells damage vulnerable young shoots. However, the metabolic signals that regulate C-repeat binding factor (CBF)-mediated cold responses remain unclear. Time-series metabolomic analysis revealed progressive glutamate accumulation in cold-treated tea shoots, accompanied by a marked decline in theanine content. Exogenous glutamate enhanced cold tolerance in tea shoots. By contrast, co-silencing of CsGGT2 and CsGGT4 attenuated cold-induced glutamate accumulation and compromised cold tolerance, indicating that CsGGT2/CsGGT4-associated theanine turnover contributes to cold-induced glutamate accumulation. Silencing CsGLR3.2 attenuated the protective effect of glutamate and reduced glutamate-enhanced CsCBF1–CsCBF4 expression. Pharmacological treatments provided complementary support for the involvement of GLR- and Ca²⁺-associated signaling in this response. Transcriptomic analysis further showed that glutamate altered the expression of genes associated with flavonoid biosynthesis and redox homeostasis. Collectively, these findings connect the characteristic theanine metabolism of tea plants with cold-responsive glutamate signaling and support a model in which glutamate enhances CsCBF expression through CsGLR3.2- and Ca²⁺-associated signaling. Glutamate metabolism and GLR-associated signaling therefore represent potential targets for improving cold tolerance in tea plants.