Identification and Functional Evaluation of Leucine in Enhancing Cold Tolerance of Tea Plant's Young Shoots

Cold stress severely impairs the growth and metabolic composition of tea shoots, thereby limiting tea production. The application of exogenous regulatory substances offers a potential strategy to mitigate cold-induced damage in tea plants. Therefore, identifying endogenous metabolites that could increase cold tolerance and exploring their working mechanisms would be of interest. Here, we identified 292 differentially accumulated metabolites in young shoots of two different tea cultivars through UPLC-MS/MS assays, among which amino acid metabolism was found to be markedly altered. Further analysis revealed that leucine was dramatically induced in all 10 tested accessions. Phenotype, relative electrolyte leakage (REL), and maximum quantum yield of photosystem II (Fv/Fm) analyses revealed that pre-treatment with 20 mM leucine significantly increased the cold tolerance of young shoots in 4 different cultivars. Transcriptome analysis revealed that 48.06% of differentially expressed genes (DEGs), including calcium (Ca²⁺) signaling, sugar conversion, and reactive oxygen species (ROS) scavenging pathways, were similarly induced by leucine pretreatment in both tolerant and sensitive cultivars. However, 51.94% DEGs involved in phenylpropanoid biosynthesis, glutathione metabolism, and tyrosine metabolism were differentially expressed. Moreover, free amino acid detection also revealed great differences among tolerant and sensitive cultivars. Taken together, our results demonstrate that exogenous application of 20 mM leucine, a cold-inducible endogenous metabolite, effectively mitigates cold-induced damage in both tolerant and sensitive cultivars through common and cultivar-specific mechanisms. These findings supported the development of leucine as a promising cold resistance agent for mitigating the effects of cold spells on young shoots in fields.