Identification And Functional Characterization Of Theanine Transporter CsAAPs Gene In Tea Plant(Camellia Sinensis L.)

Theanine is a unique non-protein amino acid,which confers the tea infusion a fresh and affects the quality and price of the tea.Therefore,it is critical to enhance the theanine content for both tea production and tea quality.Previously,it has been proved that theanine is mainly synthesized in the roots of tea trees,and then transported to the shoot through the vascular system.In tea plants,how theanine is loaded into and unloaded from the vascular bundle is still unknown.Previous studies have found that long-distance transport of amino acids are mediated by amino acid transporters.We hypothesized that the AAP family amino acid transporters with a broad spectrum of substrates could be involve in theanine transportation.By performing a series of gene expression and gene functional analyses we obtained the following findings:1)The CsAAPs and AAPs in other species are highly conserved at protein sequence level.The similarity among the different members of CsAAPs is 65.91%,and the similarity among CsAAPs and Arabidopsis thaliana AAP members is 55%-80%.2)CsAAPs could be transport theanine.we found that CsAAP1,CsAAP2,CsAAP4,CsAAP5,CsAAP6,and CsAAP8 exhibited theanine transport activity,and those Km was 0.45,0.95,0.26,0.33,0.35 and 0.57 mM,respectively.CsAAP4 has the highest affinity,and CsAAP2 has the lowest affinity,for theanine.In addition,These six theanine transporters belong to the H+-dependent amino acid transporter.3)CsAAPs expression is tissue-specific in tea plant.we found CsAAP1 mainly expressed in tender leaves and stem;CsAAP2 and CsAAP8 mainly expressed in the roots;CsAAP4 and CsAAP6 mainly expressed in the vascular system,including major veins,vascular bundles and stems;CsAAP5 was constitutively expressed in the tissues examined.4)CsAAPs expression is affected by different levels and forms of nitrogen.By treating tea plants with different nitrogen levels and forms,we observed that the CsAAPs expression was induced by N deficiency.CsAAP1,CsAAP6 and CsAAP8 were also induced by NO3-while CsAAP5 was induced by NH4+.5)CsAAP1 plays an important role in mediating the long distance transport of theanine from the root to the bud.