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 were affected by light in leaf tissue. These results enhance our knowledge of biochemistry and molecular biology of the biosynthesis of functional tea compounds.