Fig. 4.

Key genes involved in the theanine biosynthesis pathway. (A) The proposed pathway for theanine biosynthesis and expression of key genes upon precursor ethylamine feeding. TS, GS, GOGAT, GDH, and ADC represent genes encoding theanine synthetase, glutamine synthetase, glutamate synthetase, glutamate dehydrogenase, and arginine decarboxylase, respectively. Tea seedlings grown hydroponically were fed ethylamine chloride for different numbers of days before being sampled for amino acid profiling and transcriptome analyses. (B) Phylogenetic tree of tea TS and GS candidate genes and the available GS genes from prokaryotes, fungi, and plants. The tea TS candidate gene (CsTSI) shows high similarity to known GSI-type genes, and other GS candidate genes exhibit high homology with previously reported GSII-type genes in plants. (C) Assay of theanine synthesis activity of CsTSI in Arabidopsis seedlings. The candidate tea TS gene (CsTSI) that shows high similarity to known GSI-type genes was cloned into a binary vector and overexpressed in Arabidopsis driven by a 35S promoter. CsTSI-OE indicates CsTSI-overexpression lines, while WT represents wild type (control). Seedlings were fed with or without 10 mM EA chloride solution (with water as control) for 3 d. Theanine synthesized by the seedlings was extracted and measured. Data are expressed as means ± SD from at least three independent transgenic lines with replicate experiments. FW, fresh weight.