Functional Analysis Of The K~+/H~+Antiporter AtKEA Gene Family In Arabidopsis Thaliana

Na+,K+/H+antiporters are H+-coupled cotransporters whose biochemical activity is to transfer the Na+or K+across a membrane in exchange for protons (H+). Na+,K+/H+antiporters are critical for ion homeostasis and pH regulation in cells, and function in diverse cellular processes, including Na+and K+movement, salt tolerance, regulation of cell cycle and cell proliferation, vesicle trafficking and fusion, and biogenesis.In the Arabidopsis genome, the AtKEA gene family contains6members. AtKEAs are homologs of bacterial K+/H+antiporters KefB/KefC, indicating that AtKEAs may encode K+/H+antiporters in Arabidopsis, and play an important role in K+homeostasis. However, the physiological functions of the AtKEA gene family remain largely uncharacterizedIn this study, we characterized the ion transport activity, gene expression, cellular localizations and biological functions of the AtKEA gene family using a variety of methods and techniques including bioinformatics, yeast growth, RT-qPCR, GFP labeling and GUS staining. Our goal is to explore the role of the AtKEA gene family in potassium uptake, environmental stresses, cellular pH regulation and osmotic adjustment. Our study will pave the way for the understanding of the cellular mechanisms governing K+homeostasis in plants.The major results are listed below:(1) Bioinformatics analysis shows that the AtKEA family contains six genes forming two subgroups in the cladogram:AtKEAl-3and AtKEA4-6. AtKEAl and AtKEA2have a long N-terminal domain. Phylogenetic analysis finds that AtKEAs separate clearly from the clusters of AtNHXs and AtCHXs with their yeast orthologs, suggesting that AtKEAs may function distinctly from either AtNHXs or AtCHXs.(2) Protein organization analysis showed that AtKEA1andAtKEA2are comprised of a soluble N-terminal domain, a Na_H exchange domain and a C-terminal KTN domain. The long N-terminal domains of AtKEA1and AtKEA2were missed in early gene annotation. The cDNA sequences of the short version AtKEAl and AtKEA2, AtsKEAl and AtsKEA2, lacking the N-terminal domains but containing the Na_H exchange domains, have been cloned in yeast by Dr. John Ward lab. We attempted to clone the full-length cDNAs of AtKEAl and AtKEA2genes. Since the direct amplification from the Arabidopsis cDNA preparation was not successful, we used a two-step strategy. We separated the gene into two pieces by choosing a restriction enzyme site in the middle of the gene; the two pieces were cloned separately. We then combined them to get the full-length cDNA. For AtKEAl, an EcoRI site was chosen to separate the gene into two pieces of A1-C1870and T1871-A3582, and we successfully cloned the full-length cDNA. However, using the same strategy, we did not clone the full length AtKEA2gene. Yeast growth analysis shows that the full length AtKEAl is inactive in ion transport. Thus, the short versions AtKEA1and AtKEA2, AtsKEAl and AtsKEA2, were used in the ion transport assay in this study.(3) We successfully cloned the AtKEA3-6genes and used these gene sequences in ion transport assays.(4) The transport activity was analyzed by expressing the AtKEA genes in yeast mutants lacking multiple ion carriers. AtKEAs conferred resistance to high K+and hygromycin B but not to salt and Li+stress. These results suggest that AtKEA gene family are able to transfer K+, and play a role in vesicle trafficking. Moreover, AtKEAs, AtNHXs and AtCHXs may have different modes of action in facilitating K+homeostasis. AtKEAs function at high K+at pH5.8while AtNHXs function at high K+in acidic environments and AtCHXs at low K+under alkaline conditions.(5) RT-qPCR analysis showed that AtKEAs were expressed in both the shoot and root of Arabidopsis. The expression of AtKEAl, AtKEA3and AtKEA4was enhanced under low K+stress, whereas AtKEA2and AtKEA5were induced by sorbitol and ABA treatments. However, osmotic induction of AtKEA2and AtKEA5was not observed in aba2-3mutants, suggesting an ABA regulated mechanism for their osmotic response. AtKEAs’ expression may not be regulated by the SOS pathway since their expression was not affected in sos mutants.(6) Promoter-GUS analysis showed that AtKEAl and AtKEA3were expressed in leaves, flowers and the mature area of roots in Arabidopsis, indicating that AtKEAs may function in regulating the growth and development of the vegetative and reproductive organs in Arabidopsis.(7) The GFP tagging analysis showed that AtKEAs were distributed diversely in yeast, suggesting that AtKEAs may have diversified roles in growth and development in plants.(8) The Golgi localization of AtKEA3was demonstrated by both the stably transformed seedlings and the transient expression in protoplasts.In summary, AtKEAs expressed and localized diversely, and may play roles in K+homeostasis and osmotic adjustment in Arabidopsis.