Functional Characterization Of Amino Acid Transpoter Genes CsVAT1.3,CsLHT8L,CsCAT9.1,CsAAP3.1 In Camellia Sinensis

Amino acids are important metabolic substances in tea,and the levels of amino acid compositions determine the taste of tea soup.However,the level of amino acid depends on the nitrogen?N?uptake,assimilation,transport efficiency and allocation of the assimilated amino acids in tea plants.As a result,it is very important to identify genes related to the allocation of amino acids between source and sink,the nitrogen utilization efficiency,and improvement of the tea quality.So far,many progresses related with amino acid transporters have been achieved in Arabidopsis,rice,poplar and other model plants,whereas few studies were found in tea plant.Further,the molecular mechanisms of amino acid uptake and transport are of great importance to improve tea quality and nitrogen nutrient efficiency.In this study,four amino acid transporters,Cs VAT1.3,Cs LHT8L,Cs CAT9.1,and Cs AAP3.1 were identified in tea plant.Arabidopsis transgenic lines of each gene were obtained to determine their amino acid substrates and transport efficiency.Moreover,the potential function of Cs AAP3.1 was performed by revealing nitrogen allocation,level of amino acid and utilization efficiency.The main results are as follows:?1?The results indicated that Cs VAT1.3 was subcellular localizated in the endoplasmic reticulum,and was expressed at extremely low levels in the roots of tea plants.Cs VAT1.3's expression levels showed varied patterns in three cultivars with different content of amino acid.In BY1,the expression levels of Cs VAT1.3 in the six tissues,except for the mature leaves,were significantly increased upon the resupply of organic N.In ZC108,the transcript levels were significantly increased upon the resupply of optimal N.Significant changes of Cs VAT1.3's expression were found in response to organic N or optimal N.Moreover,it showed that Cs VAT1.3 transport Arg at high concentration?1 m M?specifically.?2?Cs LHT8L was located in the endoplasmic reticulum,and mainly expressed in the main vein of three categories of leaves.The expression levels of Cs LHT8L in different N treatments indicate that they were significantly inhibited in the 1^stMV,2^ndLeaf,2^ndMV,Mature Leaf and MMV in ZC108 and BY1 upon resupply three N treatments.In EC1,the expression levels of Cs LHT8L in young shoots,1^st Leaf,1^st MV,1^st Leaf and 2^ndMV,were the highest when exposed to organic N,indicating that Cs LHT8L is probably involved in the amino acid transport and allocation toward young leaves in EC1.The result showed Cs LHT8L transport Gly,Pro at high concentration?>1 m M?and Lys at low concentration?<1 m M?.?3?Cs CAT9.1 was dual-located in the cytoplasmic membrane and endoplasmic reticulum,the result showed that Cs CAT9.1 is involved in transporting 11 amino acids,including His,Cys,Ile,Leu,Met,Phe,Pro,Val,Gln,Glu and Thea.It was expressed with abundant expression in the root,leaves and main vein of leaves,and might play an important role in the amino acid transpot in tea plant.In BY1,ZC108 and EC1,the expression levels of Cs CAT9.1 were significantly inhibited upon the resupply of three N treatments It is intriguing that the transcript level of Cs CAT9.1 decreased with the increase of N levels in ZC108.?4?Cs AAP3.1 was dual-located in the cytoplasmic membrane and endoplasmic reticulum.The expression levels showed that Cs AAP3.1 expressed significantly in response to organic N in multiple tissues of three tea cultivars.It showed that Cs AAP3.1 transport theanine,as well 13 other amino acids of Arg,His,Lys,Cys,Ser,Thr,Tyr,Ile,Leu,Val,Gly,Phe and Glu.The relative levels of theanine in the Cs AAP3.1 overexpressing lines were significantly higher related to WT fed with theanine,confirming the uptake of theanine by Cs AAP3.1.In the N deficient condition?0.25 m M N?,N was preferentially allocated to young leaves and the level of amino-N in the young leaves was significantly lower in Cs AAP3.1-OE lines,in comparison with WT.No significant difference with respect to N accumulation in root between Cs AAP3.1-OE lines and WT,whereas the level of amino-N was significantly higher in Cs AAP3.1-OE lines relative to WT,indicating that Cs AAP3.1 enhances the allocation of N to young leaves and is involved in the amino N partitioning between source and sink organs to adajust to low nitrogen stress.In the optimal N condition?5 m M N?,compared with WT,the N accumulation in Cs AAP3.1-OE lines significantly decreased,the proportion of N in young leaves remained unchanged,whereas the level of amino-N was significantly increased,implying that Cs AAP3.1 might hold great promise to improve tea quality by optimizing nitrogen allocation.