Nonaqueous fractionation and overexpression of fluorescent tagged enzymes reveals the subcellular sites of L-theanine biosynthesis in tea.

L-Theanine is a specialized metabolite in the tea (Camellia sinensis) plant which can constitute over 50% of the total amino acids. This makes an important contribution to tea functionality and quality, but the subcellular location and mechanism of biosynthesis of L-theanine are unclear. Here, we identified five distinct genes potentially capable of synthesizing L-theanine in tea. Using a nonaqueous fractionation method we determined the subcellular distribution of L-theanine in tea shoots and roots and used transient expression in Nicotiana or Arabidopsis to investigate in vivo functions of L-theanine synthetase and also to determine the subcellular localization of fluorescent-tagged proteins by confocal laser-scanning microscopy. In tea root tissue, the cytosol was the main site of L-theanine biosynthesis, and cytosol-located CsTSI was the key L-theanine synthase. In tea shoot tissue, L-theanine biosynthesis occurred mainly in the cytosol and chloroplasts and CsGS1.1 and CsGS2 were most likely the key L-theanine synthases. In addition, L-theanine content and distribution was affected by light in leaf tissue. These results enhance our knowledge of biochemistry and molecular biology of the biosynthesis of functional tea compounds.