CsLHTs Amino Acid Transporters Involved In Amino Acids Uptake And Transport In Tea Plant(Camellia Sinensis)

Tea is an important leaf crop,and the amino acid content in buds and young leaves has a significant positive correlation with tea quality.Amino acids in tea leaves not only give the freshness of tea drinks,but also participate in the formation of tea aroma.Tea plant prefer using ammonium nitrogen.Once it absorbed by tea plant roots,the ammonium nitrogen will be assimilated into amino acids and loading into the xylem of roots,and transported upward under the effect of transpiration pressure.In addition,amino acids can also exchange between xylem and phloem,and finally unload to sink tissue.The existing research shows that plants can directly utilize inorganic nitrogen and organic nitrogen,such as amino acids.The process of amino acid absorption,intracellular,intercellular and among tissues transport depends on the amino acid transporters.However,the amino acid transporters responsible for this function in tea plants are not clear.Theanine is the most abundant free amino acid in tea plant.Its content is more than 60%of the total free amino acid content.Theanine is the characteristic secondary metabolite of tea plant that gives tea fresh taste and calming effect.The content of theanine in green tea is positively correlated with the quality and price of green tea.The metabolism of theanine in tea plant is affected by many factors,such as light,temperature,soil nutrient status and so on.Studies have shown that theanine is mainly synthesized in the roots of tea plants,and the synthesis is more vigorous in autumn and winter.In the spring of the next year,theanine transport to the new shoots through vascular bundles.However,the molecular mechanism of theanine transport and metabolism regulation in tea plants is still unknown.The study of model plants showed that amino acid transport in plants depended on amino acid transporters,and the transcription level of amino acid transporter genes was regulated by the concentration of amino acids inside and outside cells.Therefore,we speculate that theanine accumulation in fresh tea leaves is the comprehensive result of theanine metabolism and transport.In order to study the molecular mechanism of amino acid metabolism and transport in tea plants,we systematically studied the process of amino acid metabolism,transport and related function genes in tea plants,and the main conclusions are as follows:1.The contents of theanine in the roots and buds of 14 varieties at four time points in winter and spring(December 12,March 1,March 23,April 13)showed that the synthesis of theanine in roots mainly occurred before December 12 and March;the contents of theanine in buds gradually increased from the beginning of March,reached the highest level at the end of March,and decreased significantly after the middle of April.These results suggest that theanine in roots transport to new shoots from the beginning of March,and the transport decreased significantly after mid April.2.The expression of theanine biosynthesis related genes and the amino acid transporters in roots and buds of five varieties at the above four time points were measured,and the correlation between the expression level and theanine content was analyzed.The results showed that the synthesis of theanine was feedback regulated by the theanine content,while the transport of theanine was forward regulated by the theanine content;The expression of CsLHT was positively correlated with theanine content at four points,suggesting that CsLHT might involve in theanine transport.3.The phylogenetic tree and transmembrane domain of seven LHT family amino acid transporters in tea plant were analyzed.The activities and characteristics of CsLHT1 and CsLHT6 transporters were verified in yeast mutant.The results showed that: CsLHT1 is a high affinity theanine transporter,whose transport activity depends on the proton gradient inside and outside the cell membrane;CsLHT6 is a low affinity theanine transporter,and its transport activity has a low dependence on the proton gradient inside and outside the cell membrane.4.The content of free amino acids in the tea garden soil with and without chemical fertilizer was determined.The results showed that there were free amino acids in the tea garden soil,and its content was related to the fertilization.The content of free amino acids in the soil with high chemical fertilizer was lower,and the content of free amino acids in the soil without chemical fertilizer was higher.5.The experimental results of cultivating tea seedlings with amino acids as the only nitrogen source showed that the tea plants roots can directly absorb and utilize the free amino acids in the soil,which partly meets the nitrogen requirements of the tea trees.6.The analysis of tissue expression specificity showed that CsLHT1 and CsLHT6 were highly expressed in the roots of tea plants,and their expression was induced by exogenous amino acids;The functional complement experiment of yeast mutants showed that CsLHT1 and CsLHT6 had a wide range of substrate specificity,and could transport a variety of amino acids;Overexpression CsLHT1 and CsLHT6 in Arabidopsis showed that its absorb ability was enhanced then wild type.These results indicate that CsLHT1 and CsLHT6 play an important role in the absorption of amino acids from soil by tea roots.In conclusion,CsLHT1 and CsLHT6 are involved in the absorption and transport of amino acids in tea plants,and the transporter has wide substrate specificity and proton dependence.The difference of substrate affinity shows that CsLHT1 and CsLHT6 are non redundant in the process of amino acid transport.