Characterization Of Tea Plant Amino Acid Transporters CsLHTs And Autophagy Related Genes CsATGs Involved In Nitrogen Utilization

The tea plant is a perennial economic plant with the tender shoots as the harvestable portion.Nitrogen(N)is essential for the tea plant’s growth and the tea quality.Tea is rich in amino acids,and the content and composition determine the quality of tea.Amino acids,the N sources that can be directly absorbed and utilized by plants,and amino acid-N is the most abundant nitrogen species in plant long-distance transport and retranslocation by amino acid transporters(AATs).Autophagy,a pathway for degradation and recovery of intracellular materials with high efficiency,plays an important role in N recycling.Therefore,the research on the biological function of AATs and ATGs related with N utilization is supposed of great significance to improve N use efficiency of tea plants,and the yield and quality of tea.In this study,based on the RNA-Seq of for seven tissues in tea plants,the weighted gene co-expression network analysis(WGCNA)identified seven hub genes from N-regulatory networks,including four CsLHTs(Lysine and histidine transporters)and three from the CsATGs family.Further,the members of the CsLHTs subfamily and the CsATGs family were identified genome-wide,and the function in N utilization was performed for the seven candidate genes.The main results are as follows:1.The RNA-Seq showed that the gene expression pattern of mature leaf(ML)was significant different from the other tissues in tea plants.Based on the expression patterns of genes involved in N utilization,the seven tissues were classified into two groups,Group1 for ML,and Group 2 for the rest of the tissues,respectively.The GO enrichment terms were related with N metabolism in various nitrogen compounds metabolism processes,such as aminos,polypeptides,and amide compounds.The KEGG metabolic pathway analysis suggested that the maintenance of metabolism homeostasis in ML was related to autophagy.Additionally,based on WGCNA,seven candidate genes were identified from three Nregulatory networks,including CsLHT1,CsLHT4,CsLHT7,CsLHT11,CsATG8 a,CsATG8 f,and CsATG101.2.A total number of 22,22,17,16,12,8 and 7 CsLHTs subfamily members were identified from the genomes of Shuchazao(CSS),Yunkang 10(YK10),Biyun(BY),Tieguanyin(TGY),Longjing 43(LJ43),Huangdan(HD),and an ancient tea tree(DASZ).A genome-wide survey on CSS identified a total of 80 CsATGs,among which ATG18,ATG8 and ATG16 were the subfamilies with the most family members,with 18,12 and 8,respectively.Moreover,the protein sequences of 22 CsLHTs from CSS along with ten At LHTs from Arabidopsis were phylogenetically divided LHTs into two evolutionary clades,among which CsLHT4,CsLHT7,CsLHT11,and CsLHT12 were in the same clade as At LHT4 and At LHT7.3.The expression patterns showed that CsLHT1 was mainly expressed in roots and mature leaves of tea plants.And the expression levels of CsLHT1 were affected by both N levels and tea cultivars,among which the recovery with NSM(a mixture of inorganic and amino acid-N)induced the expression of CsLHT1 in new leaves of tea cultivar Longjing43(LJ43).Heterologous expression of CsLHT1 in yeast demonstrated that CsLHT1 participated in the absorption of theanine(Thea),but not asparagine(Asn),glutatic acid(Glu),threonine(Thr),and other amino acids.However,heterologous expression of CsLHT1 in Arabidopsis revealed that CsLHT1 could mediate the absorption of histidine(His),glycine(Gly),valine(Val),leucine(Leu),and a number of other amino acids in Arabidopsis.Additionally,the sensitive to low nitrogen of the Arabidopsis mutant Atlht1(SALK＿034566)was restored upon the expression of CsLHT1.A possible conclusion was obtained that CsLHT1 mediates the uptake or loading of amino acids into the xylem in roots,as well as the distribution in the leaves of tea plants.CsLHT1 might specifically transport Thea in tea plants,while the conserved function with At LHT1 suggests that CsLHT1 is involved in nitrogen use efficiency in plants.4.CsLHT4,CsLHT7 and CsLHT11 are hub genes identified in the Brown module of the N-regulatory network by WGCNA.They were highly expressed both in the roots and in the leaves of LJ43,indicating the mediating the uptake or loading into xylem of amino acids in roots,as well as the distribution in leaves.The expression levels of all the three genes were induced by NSM.Heterologous expression of CsLHT4,CsLHT7,and CsLHT11 in yeast showed no ability in transporting Thea,Glu,Asn,and other amino acids.However,the inhibited growth of CsLHT4,CsLHT7,and CsLHT11 overexpressing Arabidopsis showed that the three genes were likely to take up Gln,His,Thr,and some other amino acids.Under hydroponically cultured conditions,the stages from vegetative growth to reproductive growth were changed in overexpressed Arabidopsis plants compared with that in WT(wild type).Additionally,silencing CsLHT4,CsLHT7,or CsLHT11 in tea plant leaves by VIGS(Virus-induced gene silencing)decreased the expression levels of the genes related with ethylene synthesis.Together,these results suggest that CsLHT4,CsLHT7 and CsLHT11 may also be involved in the synthesis of ethylene in tea plants.5.It was found that the expression levels of CsATG8 a,CsATG8 f and CsATG101 increased with the increase in leaf maturity overall.Heterologous expression of CsATG8 a in Arabidopsis showed a significant increase in N accumulation compared with that in WT under both LN(low nitrogen)and NN(normal nitrogen),indicating CsATG8 a is involved in the improvement of N use efficiency in Arabidopsis.Heterologous expression of CsATG8 f and CsATG101 in Arabidopsis showed that compared with WT plants,overexpression of CsATG8 f or CsATG101 prolonged the transition from vegetative growth to reproductive growth.The accelerated senescence of the rosette during the reproductive growth stage was observed,regardless of N conditions for CsATG8 f overexpression Arabidopsis,but under LN for CsATG101 overexpression Arabidopsis.Together,these results indicated that both CsATG8 f and CsATG101 played roles in regulating the transition from vegetative growth to reproductive growth,suggesting great potential in maintaining vigorous vegetative growth and extending the tea picking period in tea plants.