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 ofl-theanine are unclear. Here, we identified five distinct genes potentially capable of synthesizingl-theanine in tea. Using a nonaqueous fractionation method, we determined the subcellular distribution ofl-theanine in tea shoots and roots and used transient expression inNicotianaorArabidopsisto investigatein vivofunctions ofl-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 ofl-theanine biosynthesis, and cytosol-located CsTSI was the keyl-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 keyl-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.

Automated summary

Theanine is a key amino acid in tea plants, synthesized mainly in the cytosol of roots and shoots. Its biosynthesis is regulated by specific genes and affected by light in leaf tissues, contributing significantly to the functionality and quality of tea.