Transcription factor CsHSFB2c suppresses CsTS1 and CsGS1 expression to reduce theanine biosynthesis in tea plants under heat stress

• Heat stress led to the unique accumulation pattern of theanine and catechins in tea plants: Theanine content decreased significantly diminished with the increasing temperature (peaked at 20°C), while catechins levels elevated with increasing temperature (peaked at 30°C). • RNA-seq identified a class B heat shock transcription factor CsHSFB2c, whose expression was negatively correlated with theanine content. • CsHSFB2c directly binds to the promoters of CsTS1 and CsGS1 and inhibits their transcription, thereby reducing theanine biosynthesis. Theanine content in tea plants is reduced by heat stress, but its molecular mechanism is still unclear. In this study, a temperature gradient treatment (20°C, 25°C, 30°C, 35°C) was performed to unveil the effect of heat stress on biosynthesis and accumulation of theanine. It was found that heat stress triggered metabolic alterations characterized by reduced theanine and increased catechin levels. In addition, heat stress up-regulated the expression of 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 reduced theanine levels. Consistent with these changes, silencing CsHSFB2c up-regulated the expression of CsTS1 and CsGS1 , while overexpression of CsHSFB2c resulted in their down-regulation. 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 heat resistance and quality of tea plants through molecular breeding.