Heat stress reduces theanine content in tea plants, but the underlying molecular mechanism remains unclear. In this study, a temperature gradient treatment (20℃, 25℃, 30℃, and 35℃) was performed to unveil the effect of heat stress on biosynthesis and accumulation of theanine. We found that heat stress induced metabolic changes, characterized by decreased theanine content and increased catechin levels. In addition, heat stress up-regulated the expression of the class B heat shock transcription factor gene CsHSFB2c, while significantly suppressing the transcription of key theanine biosynthetic genes CsTS1 and CsGS1. Functional studies showed that silencing CsHSFB2c increased theanine content, while its overexpression significantly decreased theanine levels. Consistent with these changes, silencing CsHSFB2c upregulated the expression of CsTS1 and CsGS1, while overexpression of CsHSFB2c downregulated their expression. Yeast one-hybrid (Y1H) and dual-luciferase reporter gene (Dual-LUC) assays showed that CsHSFB2c directly binds to the promoters of CsTS1 and CsGS1 and inhibits their expression. These results demonstrate that CsHSFB2c mediates heat-induced suppression of theanine biosynthesis by directly inhibiting the expression of CsTS1 and CsGS1. This study provides a theoretical basis for improving the heat resistance and quality of tea plants via molecular breeding.

The ATP-binding cassette (ABC) transporter is a gene superfamily in plants. ATP-binding cassette subfamily C (ABCC) protein is a multidrug resistance-associated (MRP) transporter. They play various roles in plant growth, development, and secondary metabolite transport. However, there are few studies on ABCC transporters in tea plants. In this study, genome-wide association study (GWAS) analysis of epigallocatechin gallate (EGCG) content in 108 strains of Kingbird revealed that CsABCCs may be involved in EGCG transport. We identified 25 CsABCC genes at the genomic level of the tea plant, their phylogenetic tree, gene structure, targeted miRNA and other bioinformatics were analyzed. The expression patterns of CsABCCs in eight different tissues and abiotic stress indicate that they have potential roles in regulating the growth, development, and defense of tea plants. The correlation analysis revealed that the expression of the CsABCC11 gene was closely related to the EGCG content in tea buds of 108 strains of the Kingbird, and the subcellular localization experiments in tobacco showed that CsABCC11 protein was localized on the plasma membrane. The virus-induced gene silencing (VIGS) strategy in tea plants further verified that CsABCC11 was involved in EGCG accumulation. Our study laid a foundation for studying the biological function of CsABCC and provided a new candidate molecular marker gene for further EGCG-related variety breeding, which will be of great interest to breeders.