A Single Amino-Acid Substitution in the Sodium Transporter HKT1 Associated with Plant Salt Tolerance

Abstract

A crucial prerequisite for plant growth and survival is the maintenance of potassium uptake, especially when high sodium surrounds the root zone. The Arabidopsis HIGH-AFFINITY K(+) TRANSPORTER1 (HKT1), and its homologs in other salt-sensitive dicots, contributes to salinity tolerance by removing Na(+) from the transpiration stream. However, TsHKT1;2, one of three HKT1 copies in Thellungiella salsuginea, a halophytic Arabidopsis relative, acts as a K(+) transporter in the presence of Na(+) in yeast (Saccharomyces cerevisiae). Amino-acid sequence comparisons indicated differences between TsHKT1;2 and most other published HKT1 sequences with respect to an Asp residue (D207) in the second pore-loop domain. Two additional T salsuginea and most other HKT1 sequences contain Asn (n) in this position. Wild-type TsHKT1;2 and altered AtHKT1 (AtHKT1(N-D)) complemented K(+)-uptake deficiency of yeast cells. Mutant hkt1-1 plants complemented with both AtHKT1(N) (-) (D) and TsHKT1;2 showed higher tolerance to salt stress than lines complemented by the wild-type AtHKT1 Electrophysiological analysis in Xenopus laevis oocytes confirmed the functional properties of these transporters and the differential selectivity for Na(+) and K(+) based on the n/d variance in the pore region. This change also dictated inward-rectification for Na(+) transport. Thus, the introduction of Asp, replacing Asn, in HKT1-type transporters established altered cation selectivity and uptake dynamics. We describe one way, based on a single change in a crucial protein that enabled some crucifer species to acquire improved salt tolerance, which over evolutionary time may have resulted in further changes that ultimately facilitated colonization of saline habitats.

Figure 1.

Salt-stress responses of AtHKT1;1- and TsHKT1;2-expressing plants. A, Seeds were surface-sterilized and germinated on 0.5× MS medium with or without 75 mm NaCl. Photographs were taken after one week. B, 10-d-old seedlings grown in MS medium were transferred to soil and further grown for two weeks followed by 300-mm NaCl treatment for two weeks. Photographs were taken at the end of salt treatment. D, Seeds of wild type, hkt1-1, and the transgenic lines (ProAtHKT1::AtHKT1 and ProAtHKT1::TsHKT1;2 in the hkt1-1 background) were grown in hydroponic solution for one week, followed by 20- or 30-mm NaCl treatment as indicated. Photographs were taken two weeks after the addition of salt. C and E, After salt treatment, fresh weights from plants used in (C) and (E) (30 mm NaCl) were measured. Error bars represent sds from three independent repeats (n = 30 in each repeat). Significant difference was determined by a Student’s t -test; single or double stars indicate a P-value of <0.05 or <0.01, respectively. F, Sequence comparison of HKT homologs from Arabidopsis, T. salsuginea, and T. parvula. Amino-acid sequences in the second pore-loop region (PB) and the adjacent transmembrane domain (M2B; red boxes) are aligned with the use of Clustal-W (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The conserved Gly residues in the PB region (Mäser et al., 2002a, 2002b) are indicated by asterisks. The Asp residues specific for TsHKT1;2 (D207) and TpHKT1;2 (D205) are indicated by arrows.

Figure 2.

Electrostatic potential distribution of the pore-forming regions of AtHKT1;1, TsHKT1;2, and their mutants. Representative structures of wild type-AtHKT1 (A), Mut-AtHKT1 (B), wild type-TsHKT1;2 (C), and Mut-TsHKT1;2 (D). Pore-forming regions are represented by stick models. Electrostatic potential surfaces of wild-type and Mut-AtHKT1 (E and F) and wild-type and Mut-TsHKT1;2 (G and H) are shown, indicating each pore-forming region. The protein surface structures (E, F, G, and H) are rotated 180°. Key residues in the second pore-forming and fourth transmembrane domain regions are colored yellow and gray, respectively. Na⁺ and K⁺ ions are represented by violet and green spheres, respectively. ESP: electrostatic potential distribution.

Figure 3.

Ion selectivity and accumulation of potassium content in AtHKT1 wild-type and mutated proteins. A, Positions of conserved amino acids (Asn) in AtHKT1 that were mutated. AtHKT1 contains Asn residues in the second pore-loop region and in the adjacent transmembrane domain, whereas TsHKT1;2, at the same positions contains Asp residues (red circles and boxes indicate mutation sites, whereas N and C denote N terminus and C terminus of HKT1 protein, respectively). B, The K⁺-uptake deficient yeast strain CY162 (trk1, trk2) expressing AtHKT1, AtHKT1^N211D, AtHKT1^N211D, and AtHKT1^2N2D were grown on SD medium lacking uracil and supplemented with the indicated concentration of KCl. The SD medium has an intrinsic content of 7.5 mm K⁺. TsHKT1;2 and AtKAT1 were used as negative and positive controls, respectively. C, Potassium content in the CY162 yeast strain expressing AtHKT1, AtHKT1^N211D, AtHKT1^N211D, AtHKT1^2N2D, grown in SD medium adjusted to 10-mm KCl final concentration, with error bars representing sds from three independent repeats (n = 3). EV: pYES2 empty vector.

Figure 4.

Transport characteristics of AtHKT1, TsHKT1;2, and their respective mutants. Representative current-voltage curves from X. laevis oocytes expressing AtHKT1 and TSHKT1;2 (A), and their mutants AtHKT1^N211D and TsHKT1;2^D207N (B) at increasing Na⁺-gluconate concentrations (in mm). Dotted line indicates zero current level. The voltage-clamp protocol is given in “Materials and Methods”.

Figure 5.

Current responses of AtHKT1, TsHKT1;2, and their mutants at increasing Na⁺ and K⁺ concentrations. Currents from X. laevis oocytes expressing AtHKT1 and TSHKT1;2 (A) or their mutants AtHKT1^N211D and TsHKT1;2^D207N (B) at −150 mV and variable concentrations of either Na⁺ or K⁺ (mM). Mean and sd of 15 different oocytes for the AtHKT1, TsHKT1;2, and TsHKT1;2^D207N, and nine different oocytes for the AtHKT1^N211D.

Figure 6.

Salt stress-dependent phenotypes of transgenic plants expressing ProAtHKT1:: AtHKT1 and its mutant forms in the hkt1-1 background. A, 10-d-old MS media grown seedlings of Col-gl, hkt1-1, and transgenic plants expressing ProAtHKT1::AtHKT1, ProAtHKT1::AtHKT1^N211D, ProAtHKT1::AtHKT1^N242D, and ProAtHKT1::AtHKT1^2N2D in the hkt1-1 background were transferred to soil and further grown for two weeks followed by 300-mm NaCl treatment for another two weeks as described in “Materials and Methods”. Photographs were taken at the end of salt treatment. B, Seeds of wild type, hkt1-1, and all the transgenic lines were grown in hydroponic solution for one week followed by 20- or 30-mm NaCl treatment. Photographs were taken two weeks after the addition of salt. C and D, After salt treatment, fresh weights of plants used in (A) and (B) (30 mm NaCl) were measured. Error bars represent sds from three independent repeats (n = 30). Significant difference was determined by a Student’s t-test; single or double stars indicate a P-value of <0.05 or <0.01, respectively.

Figure 7.

Na⁺ and K⁺ content in plants. Ten-days old grown seedlings of Col-gl, hkt1-1, and transgenic plants expressing ProAtHKT1::AtHKT1 or ProAtHKT1::AtHKT1^N211D in the hkt1-1 background were transferred to hydroponic cultures and further grown for two weeks followed by 100 mm NaCl for 24 h in MS medium. Na⁺ and K⁺ contents were measured by inductively coupled plasma optical emission spectroscopy. A and B, Na⁺ contents of shoots or roots. C and D, K⁺ contents of shoots or roots.