Cloning of a high-affinity K+ transporter gene PutHKT2;1 from Puccinellia tenuiflora and its functional comparison with OsHKT2;1 from rice in yeast and Arabidopsis

Abstract

A high-affinity K+ transporter PutHKT2;1 cDNA was isolated from the salt-tolerant plant Puccinellia tenuiflora. Expression of PutHKT2;1 was induced by both 300 mM NaCl and K+-starvation stress in roots, but only slightly regulated by those stresses in shoots. PutHKT2;1 transcript levels in 300 mM NaCl were doubled by the depletion of potassium. Yeast transformed with PutHKT2;1, like those transformed with PhaHKT2;1 from salt-tolerant reed plants (Phragmites australis), (i) were able to take up K+ in low K+ concentration medium or in the presence of NaCl, and (ii) were permeable to Na+. This suggests that PutHKT2;1 has a high affinity K+-Na+ symport function in yeast. Arabidopsis over-expressing PutHKT2;1 showed increased sensitivities to Na+, K+, and Li+, while Arabidopsis over-expressing OsHKT2;1 from rice (Oryza sativa) showed increased sensitivity only to Na+. In contrast to OsHKT2;1, which functions in Na+-uptake at low external K+ concentrations, PutHKT2;1 functions in Na+-uptake at higher external K+ concentrations. These results show that the modes of action of PutHKT2;1 in transgenic yeast and Arabidopsis differ from the mode of action of the closely related OsHKT2;1 transporter.

Fig. 1.

(A) Construction of plasmid pBI121 that contains PutHKT2;1 or OsHKT2;1 open reading frame driven by the CaMV 35S promoter and nptII. RB, right border; LB, left border. (B) Expression of PutHKT2;1 and OsHKT2;1 in wild-type (WT) and T₂ generation transgenic lines. Total RNA (4.5 μg) extracted from 4-week-old seedlings were analysed by RNA gel blot.

Fig. 2.

(A) Amino acid sequence alignment of PutHKT2;1 with PhaHKT2;1 (Phragmites australis, BAE44385) and OsHKT2;1 (Oryza sativa, BAB61789). The sequences were aligned by the program ClustalW. Black and grey backgrounds indicate identical residues and similar residues, respectively. Grey and black bars above the sequences indicate the putative transmembrane (M) and pore (P) domains, respectively, by the TMpred program. The conserved Gly residue is indicated by an asterisk, while the Gln270 of TaHKT2;1 which is conserved in PhaHKT2;1 and OsHKT2;1 is indicated by a black dot. Putative M1PM2 motifs (a–d) are marked. (B) Genomic Southern hybridization of PutHKT2;1. Genomic DNA (20 μg) was digested by BamHI and HindIII. Hybridization was performed overnight at 45 °C, with a digoxigenin (DIG)-labelled PutHKT2;1 cDNA probe. (C) Unrooted minimum-evolution tree of the HKT transporters. The translated sequences of known HKT genes were aligned by the ClustalW program and the tree was constructed using the MEGA 4 program (Kumar et al., 2008). Scale bar indicates 0.1 substitutions per site.

Fig. 3.

Subcellular localization of PutHKT2;1::GFP fusions. GFP fusion constructs of PutHKT2;1::GFP or control GFP were introduced into onion epidermal cells by particle bombardment, and the fluorescent signals were examined 10 h after bombardment.

Fig. 4.

(A) Expression analysis of PutHKT2;1 by means of real-time RT-PCR in roots and shoots of P. tenuiflora subjected to 300 mM NaCl (+Na), K⁺-starvation (–K), and both 300 mM NaCl and K⁺-starvation stresses (+Na, –K) for 24 h. (B) Time-course expression analysis of PutHKT2;1 in roots of P. tenuiflora. Results are expressed as means ±SE (n=3).

Fig. 5.

Growth of yeast cells transformed by empty vector pAUR123 (control) or with the vector containing PutHKT2;1, PhaHKT2;1, or OsHKT2;1 cDNA. Yeast cells were inoculated on SC/-His plates (except for K⁺-starvation treatment when the plate was a one-quarter-strength SC/-His plate) supplemented by various concentration of NaCl and KCl as indicated with serially diluted drops of yeast cells suspensions and cultured for 5 d.

Fig. 6.

(A) Concentration dependence of K⁺ uptake (upper panel) or Na⁺ influx (lower panel) in yeasts expressing HKT2;1 of P. tenuiflora, reed plant, or rice in the presence of 5, 10, 25, 50, 100 mM NaCl, and 50 μM KCl. Strain 9.3 yeast cells were transformed with empty vector pAUR123 (open diamonds) or with the vector containing PutHKT2;1 (closed squares), PhaHKT2;1 (closed triangles) or OsHKT2;1 (grey circles) cDNA. (B) K⁺/Na⁺ ratio of yeasts cells transformed with empty plasmid (open bar) or with plasmid containing PutHKT2;1 (dark-grey bar), PhaHKT2;1 (light-grey bar) or OsHKT2;1 (black bar) cDNA treated as in (A). (C) Concentration dependence of Rb⁺ uptake in yeasts expressing HKT2;1 of P. tenuiflora, reed plant, or rice. Yeast strains and symbol are indicated as in (A). Results are expressed as means ±SE (n=3).

Fig. 7.

Ion specific sensitivity of PutHKT2;1 and OsHKT2;1 over-expression lines. (A) Seven-day-old WT and transgenic seedlings grown on MS were transferred to minimal-medium (Maser et al., 2002a) supplemented with 75 mM NaCl, 75 mM KCl, and 10 mM LiCl. Seedlings were grown vertically for nine additional days before being photographed. (B) Shoot phenotype of seedling grown in (A). Bar in each picture indicates 1 cm.

Fig. 8.

Ion specific sensitivity of PutHKT2;1 and OsHKT2;1 over-expression lines by means of root length. Seedlings were grown as in Fig. 7. Results are expressed as means ±SE (n=5).

Fig. 9.

Potassium-dependent Na-sensitivity of PutHKT2;1 and OsHKT2;1 over-expression lines. (A) Seven-day-old WT and transgenic seedlings grown on MS were transferred to minimal-medium supplemented with 1.75 mM or 0.1 mM KCl and 50 mM NaCl. (B) Shoot phenotype of seedlings grown in (A). Bar in each picture indicates 1 cm. (C) Root-lengths of WT and transgenic seedlings after the indicated treatments. Results are expressed as means ±SE (n=5).

Fig. 10.

Calcium regulation on Na-sensitivity of PutHKT2;1 and OsHKT2;1 over-expression lines. Root lengths of WT and transgenic seedlings after the indicated treatments. Seven-day-old WT and transgenic seedlings grown on MS were transferred to minimal-medium supplemented with 0, 1, 3, or 10 mM CaCl₂ and 50 mM NaCl. Results are expressed as means ±SE (n=5).

Fig. 11.

Comparison of Na⁺ and K⁺ accumulation in shoots and roots between WT, PutHKT2;1, and OsHKT2;1 over-expression lines. Seven-day-old WT and transgenic seedlings grown on MS were transferred to minimal-medium supplemented with the indicated salts. Seedlings were grown vertically for 14 additional days before being harvested. For each treatment, the ion content was determined from a pool of 15 plants. Results are expressed as means ±SE (n=3). Asterisks indicate a significant difference from the WT (P <0.05) by Student's t test.