Transcription factor CsWRKY40 regulates L-theanine hydrolysis by activating theCsPDX2.1promoter in tea leaves during withering

L-Theanine is a crucial secondary metabolite in tea and positively determines the potential quality and health benefits of tea products. Previous work found the content of L-theanine decreased during withering process, while the specific mechanism is still unknown. Here, weighted gene co-expression network analysis (WGCNA) was performed based on the transcriptome data obtained previously. The key hydrolysis gene CsPDX2.1 in L-theanine metabolism and seven candidate transcription factors were screened out. Among those transcription factors, CsWRKY40 presented the strongest activation on the CsPDX2.1 promoter (373.18-fold) by binding to W box element based on the dual luciferase assay and EMSA results. Meanwhile, CsWRKY40 protein was located in the nucleoplasm, while CsPDX2.1 was found in both the nucleoplasm and cytoplasm. Furthermore, it was confirmed that the water loss of tea leaves was the critical factor affecting the contents of ABA and L-theanine by activating the expression of CsPDX2.1 and CsPDX2.1 based on the analysis of the withering model, water-retention model and water-loss model. Our results provide a new insight into revealing the regulation mechanism of L-theanine hydrolysis metabolism.