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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.

Ardie SW, Xie L, Takahashi R, Liu S, Takano T - J. Exp. Bot. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Asian Natural Environmental Science Center (ANESC), The University of Tokyo, 1-1-1 Midori-cho, Nishitokyo-shi, Tokyo 188-0002, Japan.

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.

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(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).
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fig6: (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).

Mentions: Since PutHKT2;1 had been shown to share high similarity with PhaHKT2;1 and OsHKT2;1 at the amino acid level, the function of these proteins was compared in a yeast expression system. PutHKT2;1, OsHKT2;1, and PhaHKT2;1 cDNAs were introduced and expressed in yeast strain 9.3 cells and the ion uptake characteristics of the cells were analysed. Previous functional characterization in yeasts showed that PhaHKT2;1 from the Nanpi area functions as a high affinity K+-Na+ co-transporter (Takahashi et al., 2007), while OsHKT2;1 functions as a Na+ selective uniporter (Uozumi et al., 2000). In this study, yeasts expressing PutHKT2;1 or PhaHKT2;1 grew better than the strain expressing OsHKT2;1 under micromolar K+ concentrations (10 μM K+) and were able to complement the K+ uptake deficiency phenotype, whereas the yeast strain expressing OsHKT2;1 did not (Fig. 5). Furthermore, the suppression of yeast growth by 100 mM NaCl was rescued by the addition of K+ in the yeasts expressing PutHKT2;1 and PhaHKT2;1, but not in the yeast expressing OsHKT2;1. To investigate further whether the growth promotion of yeasts expressing PutHKT2;1 and PhaHKT2;1 cDNA was due to their ability to take up K+, the ion uptake ability of those yeasts was measured. The yeasts expressing PutHKT2;1 or PhaHKT2;1 showed a high K+ uptake ability in the Rb+ uptake experiment, while the yeast expressing OsHKT2;1 did not (Fig. 6C). Similar to PhaHKT2;1, PutHKT2;1 functioned in the micromolar range of Rb+ (K+), indicating that it mediated high affinity K+ uptake. PutHKT2;1 also showed permeability to Na+ (Fig. 6A, lower panel). Increased Na+ influx with an increasing concentration of NaCl was observed in all of the transformed yeasts. However, yeasts expressing PutHKT2;1 and PhaHKT2;1 could maintain their K+ uptake ability (Fig. 6A, upper panel), and, as a result, had a higher K+/Na+ ratio than the yeast strain expressing OsHKT2;1 (Fig. 6B).


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.

Ardie SW, Xie L, Takahashi R, Liu S, Takano T - J. Exp. Bot. (2009)

(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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2724696&req=5

fig6: (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).
Mentions: Since PutHKT2;1 had been shown to share high similarity with PhaHKT2;1 and OsHKT2;1 at the amino acid level, the function of these proteins was compared in a yeast expression system. PutHKT2;1, OsHKT2;1, and PhaHKT2;1 cDNAs were introduced and expressed in yeast strain 9.3 cells and the ion uptake characteristics of the cells were analysed. Previous functional characterization in yeasts showed that PhaHKT2;1 from the Nanpi area functions as a high affinity K+-Na+ co-transporter (Takahashi et al., 2007), while OsHKT2;1 functions as a Na+ selective uniporter (Uozumi et al., 2000). In this study, yeasts expressing PutHKT2;1 or PhaHKT2;1 grew better than the strain expressing OsHKT2;1 under micromolar K+ concentrations (10 μM K+) and were able to complement the K+ uptake deficiency phenotype, whereas the yeast strain expressing OsHKT2;1 did not (Fig. 5). Furthermore, the suppression of yeast growth by 100 mM NaCl was rescued by the addition of K+ in the yeasts expressing PutHKT2;1 and PhaHKT2;1, but not in the yeast expressing OsHKT2;1. To investigate further whether the growth promotion of yeasts expressing PutHKT2;1 and PhaHKT2;1 cDNA was due to their ability to take up K+, the ion uptake ability of those yeasts was measured. The yeasts expressing PutHKT2;1 or PhaHKT2;1 showed a high K+ uptake ability in the Rb+ uptake experiment, while the yeast expressing OsHKT2;1 did not (Fig. 6C). Similar to PhaHKT2;1, PutHKT2;1 functioned in the micromolar range of Rb+ (K+), indicating that it mediated high affinity K+ uptake. PutHKT2;1 also showed permeability to Na+ (Fig. 6A, lower panel). Increased Na+ influx with an increasing concentration of NaCl was observed in all of the transformed yeasts. However, yeasts expressing PutHKT2;1 and PhaHKT2;1 could maintain their K+ uptake ability (Fig. 6A, upper panel), and, as a result, had a higher K+/Na+ ratio than the yeast strain expressing OsHKT2;1 (Fig. 6B).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Asian Natural Environmental Science Center (ANESC), The University of Tokyo, 1-1-1 Midori-cho, Nishitokyo-shi, Tokyo 188-0002, Japan.

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.

Show MeSH
Related in: MedlinePlus