TsHKT1;2, a HKT1 homolog from the extremophile Arabidopsis relative Thellungiella salsuginea, shows K(+) specificity in the presence of NaCl

Abstract

Cellular Na(+)/K(+) ratio is a crucial parameter determining plant salinity stress resistance. We tested the function of plasma membrane Na(+)/K(+) cotransporters in the High-affinity K(+) Transporter (HKT) family from the halophytic Arabidopsis (Arabidopsis thaliana) relative Thellungiella salsuginea. T. salsuginea contains at least two HKT genes. TsHKT1;1 is expressed at very low levels, while the abundant TsHKT1;2 is transcriptionally strongly up-regulated by salt stress. TsHKT-based RNA interference in T. salsuginea resulted in Na(+) sensitivity and K(+) deficiency. The athkt1 mutant lines overexpressing TsHKT1;2 proved less sensitive to Na(+) and showed less K(+) deficiency than lines overexpressing AtHKT1. TsHKT1;2 ectopically expressed in yeast mutants lacking Na(+) or K(+) transporters revealed strong K(+) transporter activity and selectivity for K(+) over Na(+). Altering two amino acid residues in TsHKT1;2 to mimic the AtHKT1 sequence resulted in enhanced sodium uptake and loss of the TsHKT1;2 intrinsic K(+) transporter activity. We consider the maintenance of K(+) uptake through TsHKT1;2 under salt stress an important component supporting the halophytic lifestyle of T. salsuginea.

Figure 1.

Sequence comparison and phylogenetic analysis of HKT homologs in T. salsuginea. A, Comparison of HKT homologs from Arabidopsis and Thellungiella species. Amino acid sequences in the second pore loop region (P_B) and the adjacent transmembrane domain (M2_B) were aligned by ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The yeast TRK1 (ScTRK1) sequence is included for comparison. The conserved Gly residues in the P_B region (Mäser et al., 2002) are indicated by the arrowhead. The Asp residues specific for TsHKT1;2 and TpHKT1;2 are highlighted with boxes. B, Unrooted minimum-evolution phylogenetic tree of protein sequences of HKT homologs with 10,000 bootstrap replicates. Accession numbers and species for all sequences are listed in Supplemental Table S1. Yeast TRK proteins (ScTRK1 and ScTRK2) are included as an outgroup. Subfamilies are as defined by Platten et al. (2006). The scale bar shows 0.2 substitutions per site. The tree was generated using MEGA5 (http://www.megasoftware.net/). [See online article for color version of this figure.]

Figure 2.

Expression of HKT homologs and construction of T. salsuginea TsHKT-RNAi transgenic plants. A and B, HKT expression patterns were analyzed with semiquantitative RT-PCR in T. salsuginea (A) and Arabidopsis (B). Two-week-old T. salsuginea and 10-d-old Arabidopsis seedlings were treated with 150 and 100 mm NaCl, respectively, for the indicated times. Compared are a T. salsuginea Shandong line transformed with the empty RNAi vector (Ts WT) and Arabidopsis Col-gl1 (At WT) and their respective mutant lines with compromised SOS1 expression (tssos1-4 and atsos1-1). Actin (ACT2) is used as a reference transcript. C, Diagrammatic representation of the TsHKT-RNAi construct. BAR, Bialaphos resistance; LB, left border; MAS3′, mannopine synthase transcriptional terminator; OCS-3′, octopine synthase transcriptional terminator; pMAS, mannopine synthase promoter; RB, right border. D, TsHKT expression was inhibited in T. salsuginea lines transformed with the TsHKT-RNAi construct. Shown are two representative TsHKT-RNAi transgenic lines (77-1 and 81-2) in comparison with the vector control (Ts WT).

Figure 3.

Salt-sensitive phenotypes of T. salsuginea TsHKT-RNAi lines. A to D, T. salsuginea plants harboring either the vector control (Ts WT) or the TsHKT-RNAi construct (lines 77-1 and 81-2) were grown on inert artificial soil and treated with no salt (A) or 300 mm NaCl (B) as described in “Materials and Methods.” C, After the salt treatment, the fresh weights were compared, with error bars representing sd values from three independent repeats (n = 30 in each repeat). D, Representative leaf sizes of Ts WT and TsHKT-RNAi lines were also compared. E and F, Root growth of Ts WT and TsHKT-RNAi seedlings under salt stress. Ten-day-old seedlings grown on 1/2 MS were transferred to K⁺-deficient medium (see “Materials and Methods”) supplemented with 100 mm NaCl. The photograph was taken after an additional 10 d of vertical growth (E). A magnification of the root hair zone is shown in F. Bars = 10 mm in A, B, D, and E. [See online article for color version of this figure.]

Figure 4.

Comparison of salt stress responses of Arabidopsis and yeast strains ectopically expressing TsHKT1;2 and AtHKT1. A to C, Four-day-old Arabidopsis seedlings overexpressing TsHKT1;2 (TsHKT1;2-OX) or AtHKT1 (AtHKT1-OX) with a 35S-CaMV promoter, in the wild-type (WT; Col-gl1) or an AtHKT1 knockout mutant (athkt1-3) background, were transferred to salt medium (Supplemental Table S4). The photograph was taken after vertical growth for 7 d (A), and root growth was measured (B) with three independent replicates (n = 30 in each repeat). A magnified image is presented in C to show the shoot phenotype. Bars = 10 mm in A and C. D, TsHKT1;1, TsHKT1;2, and AtHKT1 were ectopically expressed in the S. cerevisiae strain AXT3K (Δena1-4-Δnha1 Δnhx1), which lacks the Na⁺ efflux system. Cells transformed with a vector control and AtKAT1 were included as negative and positive controls, respectively. Yeast cells of decimal dilution were plated on AP medium containing 1 mm K⁺ (Control) and 150 mm NaCl with 1 and 10 mm KCl, respectively. [See online article for color version of this figure.]

Figure 5.

Compromised growth of T. salsuginea TsHKT-RNAi lines under K⁺-limiting conditions. A to C, T. salsuginea seedlings harboring either the vector control (Ts WT) or the TsHKT-RNAi construct (lines 77-1 and 81-2) were grown on 1/2 MS for 10 d and transferred to 1/2 MS control medium (A), K⁺-deficient medium (B), or the same medium supplemented with 10 mm KCl (C). The K⁺-deficient medium was modified from MS medium containing no KNO₃ and with KH₂PO₄ replaced with (NH₄)₂HPO₄ (see “Materials and Methods”; Supplemental Table S2). Photographs were taken after incubation in a vertical position for 15 d. D, The root growth in C is quantified. E, Arabidopsis wild type (WT) and athkt1 knockout mutants under K⁺-limiting conditions. Arabidopsis Col-gl1 wild-type plants and Arabidopsis plants harboring different alleles of athkt1 knockout mutants (athkt1-1, -3, and -4) were grown on 1/2 MS for 4 d and transferred to K⁺-deficient medium supplemented with 10 mm KCl. The photograph was taken after the transferred seedlings had been incubated in a vertical position for 15 d. F, The root growth in E is quantified. Error bars in D and F represent sd values of three repeats (n = 30). Bars = 10 mm in A, B, C, and E. [See online article for color version of this figure.]

Figure 6.

Distinct responses to K⁺-limiting conditions of Arabidopsis and yeast strains ectopically expressing TsHKT1;2 and AtHKT1. A and B, Four-day-old Arabidopsis seedlings overexpressing TsHKT1;2 (TsHKT1;2-OX) or AtHKT1 (AtHKT1-OX), on the wild-type (WT; Col-gl1) or an AtHKT1 knockout mutant (athkt1-3) background, were transferred to K⁺-deficient medium (Supplemental Table S2) supplemented with 20 mm NaCl. The photograph was taken after 7 d of incubation in a vertical position (A), and the root growth was quantified (B). Semiquantitative RT-PCR was used to select transgenic lines with comparable levels of ectopic HKT expression (A, top). C and D, The root growth of the same transgenic lines is compared with the wild type in the K⁺-deficient medium (Supplemental Table S2) supplemented with 1 mm Na⁺ and 0, 1, or 5 mm K⁺ ions. Photographs were taken as in A (C), and root growth was quantified as in B (D). Bars = 10 mm in A and C. Error bars in B and D represent sd values of three repeats (n = 30). E, Growth of yeast strain CY162 (Mat a, ura3-52, his3D200, his44-15, trkD1, trkD2::pcK64) cells harboring the vector control (pYES2), TsHKT1;1, TsHKT1;2, AtHKT1, and AtKAT1. Cells were plated in decimal dilutions on AP medium containing either 100 mm KCl (Control) or the indicated amounts of KCl and NaCl. Cells transformed with a vector control and AtKAT1 are included as negative and positive controls, respectively. [See online article for color version of this figure.]

Figure 7.

Effects of point mutations in positions 207 and 238 of the TsHKT1;2 protein. Yeast strain CY162 (Mat a, ura3-52, his3D200, his44-15, trkD1, trkD2::pcK64) cells harboring TsHKT1;2 were compared with cells expressing point mutants TsHKT1;2-1 (D207N), TsHKT1;2-2 (D238N), and TsHKT1;2-1/2 (D207N and D238N). Cells were spotted in decimal dilution on AP medium containing either 100 mm KCl (Control) or the indicated amounts of KCl and NaCl. Cells transformed with the vector control, AtHKT1, and AtKAT1 are included as controls. [See online article for color version of this figure.]