Functional Characterization Of An Amino Acid Transporter OsLHT4 In Rice

Nitrogen has different forms in soil,including inorganic and organic matter.Inorganic nitrogen mainly includes NO₃^-and NH₄⁺while organic nitrogen mainly includes amino acids and peptides.Plants can absorb both the forms in soil directly,and the inorganic nitrogen entering into roots is assimilated into amino acids in plastids through GS/GOGAT pathway.Amino acids are the dominant form of nitrogen with glutamine,asparagine,glutamate and aspartate being the predominant forms of transport in plants.Amino acids absorbed and assimilated by roots are transported to sink organs through xylem and phloem,thus promoting plant growth and development.During the short and long-distance transport processes of amino acids a variety of amino acid membrane transporters play decisive role in controlling the rate and direction of amino acids distribution.Plant amino acid transporters generally include two subfamilies,Amino Acid/Auxin Permeabilities（AAAP）and Amino Acid Polyamine Choline（APC）.AAAP family has at least six families including amino acid permeabilities（AAPs）and lysine histidine transporters（LHTs）.There are at least 85 amino acid transporters in rice genome,but the mechanism of how these amino acid transporters participate in and affect amino acid absorption,transportation from root to shoot,and transportation from source organs to sink organs is not very clear.OsAAP1,OsAAP3,OsAAP4,OsAAP5,OsAAP6,OsAAP7,OsAAP16,OsLHT1 and other reported amino acid transporters in rice belongs to the subfamilies of AAPs and LHTs.In this study,based on the microarray expression results of25 genes of AAPs and LHTs in rice,OsLHT4 was found specifically expressed in root at vegetative stage.In this study,the expression pattern and function of OsLHT4 in amino acid acquisition and distribution at both heterologous expression systems and rice were analyzed.The main results obtained are as follows:1.The analysis of bioinformatics suggests that the protein of OsLHT4 is located on the plasma membrane.Phylogenetic tree analysis of OsLHT4 and its homologus showed that the protein sequences of ZMPWZ37031 in maize and the amino acid coded by At LHT9have the highest similarities with OsLHT4.Quantitative RT-PCR analysis verified that OsLHT4 was specifically expressed in the root at vegetative stage.2.The functional complementation assay of OsLHT4 in amino acid absorption-deficient yeast 22Δ10αshowed that OsLHT4 has no amino acid absorption function in the yeast system.The functional assay of OsLHT4 in Xenopus laevis oocytes system demonstrated that expression of OsLHT4 resulted in significant efflux of glutamic acid,suggesting that OsLHT4 may be a efflux transporter of amino acids.3.The in planta analysis showed that OsLHT4 can affect the absorption of amino acids in rice.Three independent Oslht4 mutant lines were created by Crispr-Cas9 technique.The length of Oslht4 mutants’roots was shorter than that of wild-type when treated with 1m M D-type glutamate and aspartate as the only nitrogen source respectively.The amount of amino acids absorbed by roots of Oslht4 mutants was less than that of wild type when treated with 0.1 m M ¹⁵N labeled glutamate,glutamine,aspartate,asparagine for 30 min respectively.The evidence indicated that OsLHT4 could affect the absorption of amino acids in rice roots.4.The preliminary assay of OsLHT4 function in nitrogen distribution did not show significant effect on accumulation ratio of amino acids in rice.Wild type and Oslht4mutants of rice were treated with 1 m M ¹⁵N labeled glutamine and aspartate for 2 h respectively,and then nitrogen deficiency for 24 h to allow amino acids transport fully in rice.The ratio of ¹⁵N accumulation in root and shoot showed that Oslht4 mutants had no significant difference compared with that of wild type.Losing the function of OsLHT4 did not significantly change the content of amino acids in roots and shoots,indicating that OsLHT4 could not influence the distribution of amino acids in rice.5.OsLHT4 functions in altering rice phenotype.The plant height of Oslht4 mutants was shorter than that of wild type,but there was no significant difference in tiller numbers.At the athesis stage,compared with wild type,the dry weight of stems and panicles of Oslht4 mutants decreased;at the maturity,the dry weight of leaves and panicles of Oslht4mutants decreased.Calculating the ratio of vegetative and reproductive organs that accumulated nitrogen in the whole plant,compared with wild type,the vegetative organs of the Oslht4 mutants accumulated more nitrogen at the athesis and maturity,indicating that OsLHT4 affected nitrogen accumulation in different organs of rice.