Heterologous expression of CsAlaDC enhances thermotolerance through a functional ethylamine-theanine-GABA metabolic...

Theanine, a tea-enriched nonprotein amino acid, plays key roles in plant metabolism and stress adaptation. To test whether theanine biosynthesis can be reconstructed in a nontea crop and whether this metabolic network contributes to stress tolerance, we heterologously expressed the tea alanine decarboxylase gene CsAlaDC in tomato (Solanum lycopersicum cv. Micro-Tom). The resulting OE-CsAlaDC lines accumulated ethylamine and synthesized theanine without introducing a tea theanine synthase gene, demonstrating that endogenous tomato glutamine synthetase supports theanine formation. Transgenic plants exhibited distinct morphological changes, including dwarfism and dark-green leaves, while their fruits showed accelerated development, elevated levels of theanine and GABA, and improved quality-related traits such as enhanced lycopene accumulation. Under heat stress, OE-CsAlaDC plants maintained higher PSII efficiency, reduced membrane damage and reactive oxygen species accumulation, and stronger antioxidant enzyme activities than wild-type plants. Exogenous theanine further enhanced thermotolerance, promoted SlGAD1/2 expression, and increased GABA accumulation, whereas silencing SlGAD1 and SlGAD2 markedly diminished the protective effect of theanine. Exogenous ethylamine also conferred partial heat protection, but theanine showed a stronger association with GAD-dependent GABA biosynthesis. Collectively, these findings demonstrate that heterologous expression of CsAlaDC establishes a functional ethylamine-theanine metabolic branch in tomato and enhances thermotolerance through coordination with the GABA metabolic network, offering a promising strategy to improve both stress resilience and nutritional quality in crops.