Genetic selection of mutations in the high affinity K+ transporter HKT1 that define functions of a loop site for reduced Na+ permeability and increased Na+ tolerance

Potassium is an important macronutrient required for plant growth, whereas sodium (Na+) can be toxic at high concentrations. The wheat K+ uptake trans porter HKT1 has been shown to function in yeast and oocytes as a high affin ity K+-Na+ cotransporter, and as a low affinity Na+ transporter at high ext ernal Naf, A previous study showed that point mutations in HKT1, which conf er enhancement of Na+ tolerance to yeast, can be isolated by genetic select ion. Here we report on the isolation of mutations in new domains of HKT1 sh owing further large increases in Na+ tolerance. By selection in a Na+ ATPas e deletion mutant of yeast that shows a high Na+ sensitivity, new HKT1 muta nts at positions Gln-270 and Asn-365 were isolated. Several independent mut ations were isolated at the Asn-365 site. N365S dramatically increased Naf tolerance in yeast compared with all other HKT1 mutants. Cation uptake expe riments in yeast and biophysical characterization in Xenopus oocytes showed that the mechanisms underlying the Na+ tolerance conferred by the N365S mu tant were: reduced inhibition of high affinity Rb+ (K+) uptake at high Naconcentrations, reduced low affinity Na+ uptake, and reduced Na+ to K+ cont ent ratios in yeast. In addition, the N365S mutant could be clearly disting uished from less Na+-tolerant HKT1 mutants by a markedly decreased relative permeability for Na+ at high Na+ concentrations, The new mutations contrib ute to the identification of new functional domains and an amino acid in a loop domain that is involved in cation specificity of a plant high affinity K+ transporter and will be valuable for molecular analyses of Na+ transpor t mechanisms and stress in plants.