Functional Analyses Of Glutamate Dehydrogenases In Theanine Synthesis And Metabolism In Tea Plant (Camellia Sinensis L.)

Tea is one of the mostly consumed non-alcoholic beverages in the world.L-theanine,a characteristic non-protein amino acid in tea plants,is highly correlated with the taste and health benefits of tea infusion.Previous studies have shown that theanine is synthesized from glutamate and ethylamine（EA）.Glutamate contents in tea plants are regulated by glutamate dehydrogenase.Based on the transcriptomic data of roots under five nitrogen treatments and the tea plant genome data,we cloned the 3 CsGDHs gene,and explored the gene structures and expression of these genes.We also tested the enzyme activity of these CsGDHs in vitro,in tea plant and in yeast cells.The results revealed the involvement of glutamate dehydrogenases in nitrogen metabolism and theanine metabolism in tea plants.The main results are as follows.1.Three CsGDHs were clomed.They all encode conserved CsGDH protein with 411 amino acids.Based on phylogenetic analyses,they were named CsGDH2-1,CsGDH2-2and CsGDH2-3.They all highly expressed in the 1^stand 2^ndleaves of tea plant new shoot.CsGDH2-2 also expressed highly in the 6^thleaf of new shoot.Their expression in root was relatively low.2.The subcellular localization assay showed that three CsGDHs all localized in the mitochondria of tea plant cells.3.Exogenous theanine treatment on roots had little changes on CsGDHs expression in the roots.In contrast,CsGDHs expression in leaves was significantly induced by theanine treatment on the new shoots.4.The three CsGDHs could mediate glutamate synthesis and catabolism in vitro.While in tea plant roots,CsGDHs showed dominant activity in glutamate biosynthesis.5.The expression of CsGDH2-1 and CsGDH2-2 in yeast rescued the hypersensitivity of yeast gdh2 mutant to high concentration of theanine,suggesting CsGDH2-1 and CsGDH2-2 played glutamate or theanine catabolism activity in yeast.6.Enzyme activity assay showed that purified CsGDH2-1 cannot directly degrade theanine in vitro.