Theanine is a crucial indicator of tea quality, and its significance is closely tied to the economic value of tea. There have been many reports on the regulation mechanism of theanine synthesis and accumulation, but the mechanism by which gibberellin regulates theanine synthesis in tea plants is poorly understood. Previous studies have shown that the content of theanine experiences significant changes in the growth stages of tea shoots, displaying a strong correlation with gibberellin. This study confirmed that gibberellin significantly promoted the expression of the major gene of theanine synthesis, known as CsTSI. Additionally, the study identified CsWRKY71 as a transcription factor that mediated the regulation by gibberellin of theanine synthesis in tea plants. CsWRKY71 was localized in the nucleus and had a typical WRKY domain. It was a member of subclass IIC and its expression was significantly suppressed following exogenous GA₃ treatment. Further assays, such as the electrophoretic mobility shift assay, dual luciferase and asODN (antisense oligodeoxynucleotide) interference, demonstrated that CsWRKY71 significantly interacted with the promoter of CsTSI, which inhibited theanine synthesis by binding to the cis-acting element (C/T)TGAC(T/C) of the CsTSI promoter. Overall, the addition of exogenous gibberellin alleviated the inhibition of CsTSI by down-regulating the expression of CsWRKY71, ultimately facilitating the rapid biosynthesis of theanine. This study elucidated the molecular mechanism of CsWRKY71-mediated gibberellin regulation of theanine synthesis in tea plant. The findings not only enhance our understanding of the regulatory processes involved in theanine synthesis in tea plants, but also provide important references for maintaining the characteristics of high theanine in the tea plant.

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