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.

中文翻译：

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CsMYB213-CsbHLH35 模块调节茶树 （Camellia sinensis L.） 中的茶氨酸生物合成

茶氨酸是一种积累在茶树叶中的非无蛋白氨基酸，对茶独特的鲜味和甜味以及多种人类健康益处做出了重大贡献。然而，它在茶树叶片中的生物合成和转录调控仍有待完全了解。在这里，我们报道了 R2R3-MYB CsMYB213 和 bHLH CsbHLH35 共同调节两个关键的茶氨酸生物合成基因 CsTSI 和 CsAlaDC，并响应氮的可用性调节茶树叶片中的茶氨酸生物合成。通过将对大量转录组数据进行广泛的共表达分析，并通过酵母单杂交、凝胶转移、双荧光素酶报告基因测定以及反义抑制实验进行验证，我们证明了 CsMYB213 与 CsTSI 和 CsAlaDC 的启动子结合，并在体外和体内激活茶氨酸生物合成.虽然 CsbHLH35 单独没有表现出反式激活活性，但可以与 CsMYB213 相互作用并增强 CsMYB213 对 CsTSI 和 CsAlaDC 表达的反式激活。VIGS 实验显示，沉默茶叶中的 CsMYB213 或 CsbHLH35 可显著抑制 CsTSI 和 CsAlaDC 的表达。CsMYB213 和 CsbHLH35 均通过绿肥、氮肥单独施用或与有机肥配施而上调，与增强的茶氨酸积累一致。CsMYB213-CsbHLH35 模块在茶树中作为茶氨酸生物合成的氮响应性转录激活剂发挥作用。 本研究揭示了氮素营养下茶树茶氨酸生物合成的复杂调控机制，为提高茶叶中茶氨酸含量提供了有效的栽培策略。