The CsWRKY50-CsREM1-CsTSⅠ module inhibits theanine biosynthesis in tea plants under drought stress.

Drought affects theanine biosynthesis in tea (Camellia sinensis L.) plants, but how drought stress affects the associated regulatory mechanisms remains unclear. Here, we explored the molecular regulatory network governing theanine biosynthesis under drought stress. Prolonged drought stress significantly reduced theanine content and the expression of C. sinensis theanine synthase Ⅰ (CsTSⅠ) in tea plant leaves. We employed yeast 1-hybrid (Y1H) screening using the CsTSⅠ promoter as bait to identify transcription factors regulating CsTSⅠ transcription. Analysis of the drought stress transcriptome facilitated identification of the transcription factor C. sinensis REPRODUCTIVE MERISTEM 1 (CsREM1), whose encoding gene had Fragments Per Kilobase of exon model per Million mapped fragments values significantly correlated with theanine content and CsTSⅠ expression. Further experiments confirmed that CsREM1 can directly bind to the promoter region of CsTSⅠ, thereby positively regulating its transcription and enhancing theanine biosynthesis. To investigate the molecular mechanisms by which drought conditions inhibit theanine biosynthesis, we employed Weighted Gene Co-expression Network Analysis (WGCNA) and identified the transcription factor CsWRKY50. Our findings indicate that as the duration of drought stress increases, CsWRKY50 expression is upregulated. CsWRKY50 directly interacts with the promoter region of CsREM1, negatively regulating its transcription and, consequently, its effect on CsTSI. Ultimately, downregulation of CsTSⅠ transcription leads to diminished theanine biosynthesis. Overall, our findings indicate that the CsWRKY50-CsREM1-CsTSⅠ module is crucial for regulating theanine biosynthesis under drought stress.