A dual-limitation mechanism governs ethylamine supply for theanine biosynthesis in tea plants

L-Theanine is a characteristic non-proteinogenic amino acid in tea plants (Camellia sinensis) that determines tea quality. Its biosynthesis depends on ethylamine supply, but the specific source of ethylamine and the fine regulatory mechanisms remain unclear. In this study, we integrated short-term low-sugar treatments (stLS), virus-induced gene silencing (VIGS), transient expression in tobacco, genetic transformation of tea callus, and subcellular localization analyses to investigate the regulatory roles of alanine supply, alanine decarboxylase (CsAlaDC), and S-adenosylmethionine decarboxylase (CsSAMDC). stLS treatment significantly reduced alanine levels in callus and caused concomitant decreases in ethylamine and theanine, confirming that alanine supply is a key upstream limiting factor. Notably, VIGS-mediated silencing of alanine aminotransferase (CsALT) caused fluctuations in alanine content, suggesting complex metabolic compensation. Heterologous expression in tobacco demonstrated that both CsAlaDC and CsSAMDC could supply ethylamine for CsTS to promote theanine accumulation, whereas serine decarboxylase (CsSDC) did not. In tea callus, silencing CsAlaDC drastically reduced ethylamine and impaired theanine biosynthesis, while its overexpression promoted ethylamine accumulation but did not trigger substantial theanine production. In other words, the bottleneck shifts to 'downstream limitations'. In contrast, CsSAMDC silencing showed no significant difference from the wild type. Subcellular localization showed that CsTS was predominantly distributed in the cytoplasm. Collectively, this study proposes a dual-limitation model for theanine biosynthesis, revealing a coordinated regulatory mechanism involving precursor supply and metabolic pathway activity.