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.