Transcription factor CsWRKY40 regulates L-theanine hydrolysis by activating the CsPDX2.1 promoter 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 that the content of L-theanine decreased during the withering process, although the specific mechanism is still unknown. Here, weighted gene co-expression network analysis (WGCNA) was performed based on transcriptome data obtained previously. The key hydrolysis gene CsPDX2.1 in L-theanine metabolism and seven candidate transcription factors were screened out. Among these transcription factors, CsWRKY40 displayed the strongest activation on the CsPDX2.1 promoter (373.18-fold) by binding to the W box element, based on dual luciferase assay and EMSA results. The CsWRKY40 protein was located in the nucleoplasm, whereas CsPDX2.1 was found in both the nucleoplasm and cytoplasm. Analysis of withering, water-retention, and water-loss treatments confirmed that water loss from tea leaves was the critical factor that affected ABA and L-theanine contents by activating the expression of CsWRKY40 and CsPDX2.1. Our results provide a new insight into the regulatory mechanism of L-theanine hydrolysis metabolism.