A Shading-Responsive bHLH Transcription Factor (CsPIF7) Promotes Theanine Biosynthesis in Tea Plants (Camellia sinensis)

Shading is a widely applied strategy to increase theanine and chlorophyll levels in tea leaves during matcha production. However, the regulatory mechanism of theanine accumulation under shading conditions remains elusive. Through visualization analysis of PIF genes under shading conditions, we identified a homologue of AtPIF7 gene, CsPIF7, highly responsive to shading treatment. Through co-expression analysis, we found that CsPIF7 expression patterns exhibited a high correlation with those of the key theanine biosynthesis genes, CsTSⅠ and CsGSⅠ. Silencing of CsPIF7 significantly reduced the expression of CsTSⅠ and CsGSⅠ and theanine content. On the contrary, overexpression of CsPIF7 increased the expression levels of CsTSⅠ and CsGSⅠ and theanine content. Yeast one-hybrid (Y1H), dual-luciferase reporter assay and Electrophoretic Mobility Shift Assay (EMSA) further demonstrated that CsPIF7 could bind to the promoters of CsTSⅠ and CsGSⅠ, and activate the expression of CsTSⅠ and CsGSⅠ. In conclusion, CsPIF7 could mediate the shading-induced biosynthesis of theanine by directly binding to and activating the expression of CsTSⅠ and CsGSⅠ. This study provides new insights into molecular mechanisms for the production of greener and fresher matcha under shading conditions.