CsMYB213-CsbHLH35 module regulates theanine biosynthesis in tea plants (Camellia sinensis L.)

Theanine is a non-protein free amino acid accumulated in tea plant leaves and makes a major contribution to the unique umami and sweet taste and a multitude of human health benefits of tea. However, its biosynthesis and transcriptional regulation in tea plant leaves remain to be fully understood. Here, we report that an R2R3-MYB CsMYB213 and a bHLH CsbHLH35 co-regulated two key theanine biosynthesis genes, CsTSI and CsAlaDC, and regulate the theanine biosynthesis in tea plant leaves in response to nitrogen availability. By combining extensive co-expression analyses on a large body of transcriptomic data with validation through yeast one-hybrid, gel shift, dual-luciferase reporter assays as well as antisense inhibition experiments, we demonstrated that CsMYB213 bound to the promoters of CsTSI and CsAlaDC and activated theanine biosynthesis in vitro and in vivo. Although showing no transactivation activity alone, CsbHLH35 could interact with CsMYB213 and enhance the CsMYB213 transactivation of CsTSI and CsAlaDC expression. VIGS experiments revealed that silencing either CsMYB213 or CsbHLH35 in tea leaves significantly repressed CsTSI and CsAlaDC expression. Both CsMYB213 and CsbHLH35 were up-regulated by application of green manure, nitrogen fertilizer alone or in combination with organic fertilizer, aligning with the enhanced theanine accumulation. The CsMYB213-CsbHLH35 module functions as a nitrogen-responsive transcriptional activator of theanine biosynthesis in tea plants. The study sheds light on the complex regulatory mechanism underlying theanine biosynthesis in tea plants under nitrogen nutrition, offering effective cultivation strategies to enhance theanine content in tea leaves.