Amino Acid Transporters CsCAT2 And CsCAT6 Mediated Theanine Transport In Tea Plant(Camellia Sinensis L.)

Tea plant is an important economic crop.The new shoots of tea plants are harvested for tea production.Therefore,tea plants have a high demand of nitrogen for the growth of new shoots.Theanine is the most abundant non-protein amino acid,and is the main storage and transport form of nitrogen in tea plant.Its synthesis and transport play an important role in the growth of new shoots and the formation of tea quality.The transport of amino acids requires the mediation of amino acid transporters,but there are few studies on theanine transporters in tea plants.Based on the research of model plants and the screening results of theanine transporters in yeast,we speculate that CsCAT2 and CsCAT6 transport theanine in tea plant.We cloned CsCAT2 and CsCAT6 and studied their function and molecular mechanism of mediating theanine transport in tea plant.The main findings are as follows.(1)The results of phylogenetic tree analysis,transmembrane domains and conservative analysis indicated that CsCAT2 and CsCAT6 are conserved in sequence and structure with homologous proteins in other species.(2)The analysis of CsCAT2 and CsCAT6 expression patterns in 9 tissues of tea plant showed that CsCAT2 expressed constitutively.While CsCAT6 expressed highly tissue-specifically and expressed significantly higher in leaves than in other tissues.(3)The phenotype analysis results of CsCAT2 and CsCAT6 in yeast amino acid transport mutants showed that CsCAT2 and CsCAT6 had theanine transport activity.The dynamics analysis of theanine transport showed that CsCAT2 and CsCAT6 they are medium-affinity theanine transporters.(4)Functional analysis of CsCAT2 and CsCAT6 in yeast amino acid transport mutants and substrate competition experiments showed that CsCAT2 and CsCAT6 can also transport neutral amino acids such as glutamine andγ-aminobutyric acid,and acidic amino acids aspartic acid and glutamate,indicating that they have broad substrate specificity.(5)The analysis of transport activity at different p H values and the experimental results of H~+pump inhibitor treatment showed that CsCAT2 and CsCAT6 had high transport activity at acidic p H,and H~+pump inhibitor treatment inhibited their transport activity.This indicates that their transport activity is H~+-dependent.(6)The expression of CsCAT2 and CsCAT6 responded to different nitrogen forms and nitrogen deficiency stress.Nitrogen deficiency and nitrate nitrogen induce the expression of CsCAT2 and CsCAT6.This result suggested that CsCAT2 and CsCAT6 play a role in amino acid transport and nitrogen allocation in tea plants under nitrogen deficiency stress.(7)The results of the expression analyses of CsCAT2 and CsCAT6 in the roots and apical buds of four different varieties in winter and spring at four time points(December12,March 1,March 23,and April 13)showed that the expression of CsCAT2 in roots had a significant opposite trend with the theanine root-to-bud transport,with a negative correlation coefficient of-0.897(p<0.05),implying that CsCAT2 negatively regulates the long distance transport of theanine from the root to the bud.Taken together,these findings indicate that CsCAT2 and CsCAT6 in tea plants are medium-affinity theanine transporters.CsCAT6 may mainly mediate theanine transport in leaves,while CsCAT2 may mediate theanine transport in various tissues.Of particular importance is that CsCAT2 negatively regulates the long distance transport of theanine from the root to the bud.