Functional Analysis Of The Na~+,K~+/H~+ Antiporters AtNHX5 And AtNHX6 In Arabidopsis Thaliana

Na+,K+/H+ antiporters(NHXs)catalyze the exchange of Na+ or K+ for protons(H+).The plant Na+,K+/H+ antiporters play significant roles in various physiological processes,including ion homeostasis,pH regulation,cell expansion,vesicle trafficking,protein storage,osmotic adjustment,stress responses as well as growth and development.The Arabidopsis Na+,K+/H+ antiporter gene family contains 8 members that are divided into three distinct classes:plasma membrane NHXs,vacuolar NHXs,and endosomal NHXs.Currently,the plasma membrane NHXs and vacuolar NHXs have been studied extensively.AtNHX5 and AtNHX6 which are localized in the Golgi and TGN belong to the endosomal NHXs.The function and underlying mechanisms of AtNHX5 and AtNHX6 remain largely unclear.In this study,we characterized the tissue expression,subcellular localization and ion transport of AtNHX5 and AtNHX6 as well as their roles in K+ transport,pH regulation and growth and development using cell biology,molecular biology and genetics technology.Our goal is to understand the function and underlying mechanisms of AtNHX5 and AtNHX6 in K+ transport,pH regulation and growth and development.The major findings are listed below:(1)We first generated nhx5 nhx6 double mutant.The double knockout lines were produced by crossing nhx5 with nhx6 single mutants.The nhx5 nhx6 double mutant showed profound defects in growth and development.nhx5 nhx6 double mutants had smaller rosettes and shorter seedlings,was flowering and bolting late,and produced less seeds,indicating that AtNHX5 and AtNHX6 play an important role in growth and development.To verify the function of the AtNHX5 and AtNHX6 genes,we performed genetic complementation test.AtNHX5 and AtNHX6 genes were introduced into the nhx5 nhx6 double mutants.Transformation of nhx5 nhx6 with either AtNHX5 or AtNHX6 rescued the nhx5 nhx6 phenotype,and the growth and development of the transgenic seedlings resembled the wild-type seedlings.(2)GUS staining shows that AtNHX5 and AtNHX6 are preferentially expressed in cotyledons,hypocotyls and flowers.Both the transiently expressed Arabidopsis protoplasts and stably transformed Arabidopsis seedlings show that AtNHX5 and AtNHX6 are localized to Golgi and TGN.(3)The transport activity was analyzed by expressing the AtNHX5 and AtNHX6 genes in yeast mutants lacking multiple ion carriers.AtNHX5 and AtNHX6 conferred resistance to high K+,salt and hygromycin B stress.These results suggest that AtNHX5 and AtNHX6 are able to transfer K+ and Na+,and play a role in vesicle trafficking.Moreover,AtNHX5 and AtNHX6 and AtCHXs may have different modes of action in facilitating K+ homeostasis.AtNHX5 and AtNHX6 function at high K+ in acidic environments and AtCHXs at low K+ under alkaline conditions.(4)nhx5 nhx6 double mutants were sensitive to low K+ treatment and contained less K+;however,overexpression of either the AtNHX5 or AtNHX6 gene in nhx5 nhx6 recovered root growth to the wild-type levels,implying their crucial roles in K+homeostasis.(5)AtNHX5 and AtNHX6 contain four conserved acidic amino acids in transmembrane domains when aligned with the ScNhx1p sequence:D164,E188,D193 and E320 in AtNHX5;D165,E189,D194 and E320 in AtNHX6.Yeast growth analysis showed that mutations of three of the four conserved acidic amino acids(the D164 N,E188 Q and D193 N mutants of AtNHX5,and the D165N,E189Q and D194 N mutants of AtNHX6)failed to recover yeast growth in high K+ and hygromicin B.To further test the function of the conserved acidic residues in growth and development in Arabidopsis,we expressed AtNHX5 and AtNHX6 genes mutated in these four conserved residues in nhx5 nhx6 double mutant seedlings.Consistent with the yeast test,mutations of these three acidic amino acids in both AtNHX5 and AtNHX6 failed to complement the growth of the nhx5 nhx6 seedlings.These results suggest that the conserved acidic amino acids play essential roles in ion transport as well as growth and development in Arabicdopsis.(6)The pH of both the vacuoles and cell sap of nhx5 nhx6 double mutants were reduced compared with that of the wide-type seedlings,indicating that AtNHX5 and AtNHX6 regulate cellular pH homeostasis.In summary,the cellular K+ and H+ homeostasis maintained by the endosomal Na+,K+/H+ antiporters is crucial for plant growth and development.Three conserved acidic residues are essential for K+ Transport.