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Functional characterization of a wheat NHX antiporter gene TaNHX2 that encodes a K(+)/H(+) exchanger.

Xu Y, Zhou Y, Hong S, Xia Z, Cui D, Guo J, Xu H, Jiang X - PLoS ONE (2013)

Bottom Line: However, the expression of TaNHX2 did not affect the sodium concentration in transgenic cells.Our data suggest that TaNHX2 is a endomembrane-bound protein and may primarily function as a K(+)/H(+) antiporter, which is involved in cellular pH regulation and potassium nutrition under normal conditions.Under saline conditions, the protein mediates resistance to salt stress through the intracellular compartmentalization of potassium to regulate cellular pH and K(+) homeostasis.

View Article: PubMed Central - PubMed

Affiliation: College of Agronomy/Key laboratory of Physiological Ecology and Genetic Improvement of Food Crops in Henan Province, Henan Agricultural University, Zhengzhou, China.

ABSTRACT
The subcellular localization of a wheat NHX antiporter, TaNHX2, was studied in Arabidopsis protoplasts, and its function was evaluated using Saccharomyces cerevisiae as a heterologous expression system. Fluorescence patterns of TaNHX2-GFP fusion protein in Arabidopsis cells indicated that TaNHX2 localized at endomembranes. TaNHX2 has significant sequence homology to NHX sodium exchangers from Arabidopsis, is abundant in roots and leaves and is induced by salt or dehydration treatments. Western blot analysis showed that TaNHX2 could be expressed in transgenic yeast cells. Expressed TaNHX2 protein suppressed the salt sensitivity of a yeast mutant strain by increasing its K(+) content when exposed to salt stress. TaNHX2 also increased the tolerance of the strain to potassium stress. However, the expression of TaNHX2 did not affect the sodium concentration in transgenic cells. Western blot analysis for tonoplast proteins indicated that the TaNHX2 protein localized at the tonoplast of transgenic yeast cells. The tonoplast vesicles from transgenic yeast cells displayed enhanced K(+)/H(+) exchange activity but very little Na(+/)H(+) exchange compared with controls transformed with the empty vector; Na(+)/H(+) exchange was not detected with concentrations of less than 37.5 mM Na(+) in the reaction medium. Our data suggest that TaNHX2 is a endomembrane-bound protein and may primarily function as a K(+)/H(+) antiporter, which is involved in cellular pH regulation and potassium nutrition under normal conditions. Under saline conditions, the protein mediates resistance to salt stress through the intracellular compartmentalization of potassium to regulate cellular pH and K(+) homeostasis.

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Comparison of yeast cell growth upon hygromycin B treatment.Serial dilutions of the strains were grown on YPD plates containing 0 (control), 30 and 50 µg/ml hygromycin B. Δvnx1: vnx1 mutant yeast strain; TaNHX2: Δnhx1 and Δvnx1 double mutant strain OC02 transformed with plasmid pDR195-TaNHX2; Δnhx1 Δvnx1: Δnhx1 and Δvnx1 double mutant strain OC02 transformed with the empty plasmid pDR195.
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pone-0078098-g007: Comparison of yeast cell growth upon hygromycin B treatment.Serial dilutions of the strains were grown on YPD plates containing 0 (control), 30 and 50 µg/ml hygromycin B. Δvnx1: vnx1 mutant yeast strain; TaNHX2: Δnhx1 and Δvnx1 double mutant strain OC02 transformed with plasmid pDR195-TaNHX2; Δnhx1 Δvnx1: Δnhx1 and Δvnx1 double mutant strain OC02 transformed with the empty plasmid pDR195.

Mentions: Cation/H+ antiporters NHX1 and VNX1 are responsible for the cation/H+ exchange in prevacuolar and vacuolar membranes of yeast cells; therefore, an nhx1 and vnx1 double mutant yeast strain lacking monovalent cation/H+ exchange activity represents a powerful tool for the study of heterologous expression and functional characterization of endosomal monovalent cation/H+ antiporters [27]. Yeast NHX1 is a pre-vacuolar-bound Na+/H+ antiporter that mediates cation/H+ exchange in prevacuoles and possibly in other intracellular compartments; however, its Na+ (K+)/H+ exchange activity could also be detected in tonoplast vesicles isolated from an ScNHX1-transgenic Δnhx1 and Δvnx1 yeast strain OC02, which is almost devoid of backgrounds of K+/H+ and Na+/H+ exchanges at the tonoplast [27], suggesting that ScNHX1 is localized at the tonoplast in ScNHX1 transgenic yeast cells. Growth tests on plates containing hygromycin B showed that TaNHX2 complemented the lack of endogenous ScNHX1 and could increase significantly the tolerance of transgenic yeast cells to hygromycin B stress comparing with control cells (Figure 7). This result is similar to that obtained using its counterparts from other plants, which were localized in pre-vacuolar membranes/tonoplast, like ScNHX1 [18], [33]. This indicates that TaNHX2 may have similar subcellular distribution in transgenic yeast cells as AtNHX1 and ScNHX1, and may be located at the tonoplast in transgenic yeast cells. To test this hypothesis, the histidine tagged TaNHX2 (TaNHX2-His) gene was transformed into the yeast strain OC02, the vacuolar membranes were isolated from untransformed yeast cells and from TaNHX2-His transgenic yeast cells and the tonoplast proteins were analyzed by western blotting with an anti-histidine antibody. Figure 8 shows that a band with a molecular weight of about 50 KDa was observed in transgenic yeast cells, which is similar to the protein detected using the total membrane fraction (Fig. 4); however, a signal was not detected in the tonoplast fraction from untransformed yeast cells. These results indicated that TaNHX2 is located at the tonoplast membrane in the transgenic yeast cells. To test whether TaNHX2 functions as a cation/H+ exchanger like its counterparts AtNHX1 and ScNHX1 in transgenic yeast cells [27], we isolated tonoplast vesicles from yeast strain OC02 transformed with empty vector pDR195 and TaNHX2-transgenic yeast strain OC02. In the present work, little backgrounds of K+/H+ and Na+/H+ exchange activity were observed in the vacuolar membranes of yeast strain OC02 with double mutations of nhx1 and vnx1 (Fig. 9A and B). The tonoplast vesicles from transgenic yeast expressing TaNHX2 exhibited a low Na+/H+ and higher K+/H+ exchange activities (Fig. 9A, B and C). Even in absolute terms, that is, subtraction of the backgrounds of Na+(K+)/H+ exchange in vacuolar membrane of yeast cells transformed with empty vector pDR195, K+/H+ exchange activity was about two times more than Na+/H+ exchange activity in vesicles derived from yeast cells expressing TaNHX2. These results suggest that TaNHX2 functions mainly as a K+/H+ antiporter.


Functional characterization of a wheat NHX antiporter gene TaNHX2 that encodes a K(+)/H(+) exchanger.

Xu Y, Zhou Y, Hong S, Xia Z, Cui D, Guo J, Xu H, Jiang X - PLoS ONE (2013)

Comparison of yeast cell growth upon hygromycin B treatment.Serial dilutions of the strains were grown on YPD plates containing 0 (control), 30 and 50 µg/ml hygromycin B. Δvnx1: vnx1 mutant yeast strain; TaNHX2: Δnhx1 and Δvnx1 double mutant strain OC02 transformed with plasmid pDR195-TaNHX2; Δnhx1 Δvnx1: Δnhx1 and Δvnx1 double mutant strain OC02 transformed with the empty plasmid pDR195.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815223&req=5

pone-0078098-g007: Comparison of yeast cell growth upon hygromycin B treatment.Serial dilutions of the strains were grown on YPD plates containing 0 (control), 30 and 50 µg/ml hygromycin B. Δvnx1: vnx1 mutant yeast strain; TaNHX2: Δnhx1 and Δvnx1 double mutant strain OC02 transformed with plasmid pDR195-TaNHX2; Δnhx1 Δvnx1: Δnhx1 and Δvnx1 double mutant strain OC02 transformed with the empty plasmid pDR195.
Mentions: Cation/H+ antiporters NHX1 and VNX1 are responsible for the cation/H+ exchange in prevacuolar and vacuolar membranes of yeast cells; therefore, an nhx1 and vnx1 double mutant yeast strain lacking monovalent cation/H+ exchange activity represents a powerful tool for the study of heterologous expression and functional characterization of endosomal monovalent cation/H+ antiporters [27]. Yeast NHX1 is a pre-vacuolar-bound Na+/H+ antiporter that mediates cation/H+ exchange in prevacuoles and possibly in other intracellular compartments; however, its Na+ (K+)/H+ exchange activity could also be detected in tonoplast vesicles isolated from an ScNHX1-transgenic Δnhx1 and Δvnx1 yeast strain OC02, which is almost devoid of backgrounds of K+/H+ and Na+/H+ exchanges at the tonoplast [27], suggesting that ScNHX1 is localized at the tonoplast in ScNHX1 transgenic yeast cells. Growth tests on plates containing hygromycin B showed that TaNHX2 complemented the lack of endogenous ScNHX1 and could increase significantly the tolerance of transgenic yeast cells to hygromycin B stress comparing with control cells (Figure 7). This result is similar to that obtained using its counterparts from other plants, which were localized in pre-vacuolar membranes/tonoplast, like ScNHX1 [18], [33]. This indicates that TaNHX2 may have similar subcellular distribution in transgenic yeast cells as AtNHX1 and ScNHX1, and may be located at the tonoplast in transgenic yeast cells. To test this hypothesis, the histidine tagged TaNHX2 (TaNHX2-His) gene was transformed into the yeast strain OC02, the vacuolar membranes were isolated from untransformed yeast cells and from TaNHX2-His transgenic yeast cells and the tonoplast proteins were analyzed by western blotting with an anti-histidine antibody. Figure 8 shows that a band with a molecular weight of about 50 KDa was observed in transgenic yeast cells, which is similar to the protein detected using the total membrane fraction (Fig. 4); however, a signal was not detected in the tonoplast fraction from untransformed yeast cells. These results indicated that TaNHX2 is located at the tonoplast membrane in the transgenic yeast cells. To test whether TaNHX2 functions as a cation/H+ exchanger like its counterparts AtNHX1 and ScNHX1 in transgenic yeast cells [27], we isolated tonoplast vesicles from yeast strain OC02 transformed with empty vector pDR195 and TaNHX2-transgenic yeast strain OC02. In the present work, little backgrounds of K+/H+ and Na+/H+ exchange activity were observed in the vacuolar membranes of yeast strain OC02 with double mutations of nhx1 and vnx1 (Fig. 9A and B). The tonoplast vesicles from transgenic yeast expressing TaNHX2 exhibited a low Na+/H+ and higher K+/H+ exchange activities (Fig. 9A, B and C). Even in absolute terms, that is, subtraction of the backgrounds of Na+(K+)/H+ exchange in vacuolar membrane of yeast cells transformed with empty vector pDR195, K+/H+ exchange activity was about two times more than Na+/H+ exchange activity in vesicles derived from yeast cells expressing TaNHX2. These results suggest that TaNHX2 functions mainly as a K+/H+ antiporter.

Bottom Line: However, the expression of TaNHX2 did not affect the sodium concentration in transgenic cells.Our data suggest that TaNHX2 is a endomembrane-bound protein and may primarily function as a K(+)/H(+) antiporter, which is involved in cellular pH regulation and potassium nutrition under normal conditions.Under saline conditions, the protein mediates resistance to salt stress through the intracellular compartmentalization of potassium to regulate cellular pH and K(+) homeostasis.

View Article: PubMed Central - PubMed

Affiliation: College of Agronomy/Key laboratory of Physiological Ecology and Genetic Improvement of Food Crops in Henan Province, Henan Agricultural University, Zhengzhou, China.

ABSTRACT
The subcellular localization of a wheat NHX antiporter, TaNHX2, was studied in Arabidopsis protoplasts, and its function was evaluated using Saccharomyces cerevisiae as a heterologous expression system. Fluorescence patterns of TaNHX2-GFP fusion protein in Arabidopsis cells indicated that TaNHX2 localized at endomembranes. TaNHX2 has significant sequence homology to NHX sodium exchangers from Arabidopsis, is abundant in roots and leaves and is induced by salt or dehydration treatments. Western blot analysis showed that TaNHX2 could be expressed in transgenic yeast cells. Expressed TaNHX2 protein suppressed the salt sensitivity of a yeast mutant strain by increasing its K(+) content when exposed to salt stress. TaNHX2 also increased the tolerance of the strain to potassium stress. However, the expression of TaNHX2 did not affect the sodium concentration in transgenic cells. Western blot analysis for tonoplast proteins indicated that the TaNHX2 protein localized at the tonoplast of transgenic yeast cells. The tonoplast vesicles from transgenic yeast cells displayed enhanced K(+)/H(+) exchange activity but very little Na(+/)H(+) exchange compared with controls transformed with the empty vector; Na(+)/H(+) exchange was not detected with concentrations of less than 37.5 mM Na(+) in the reaction medium. Our data suggest that TaNHX2 is a endomembrane-bound protein and may primarily function as a K(+)/H(+) antiporter, which is involved in cellular pH regulation and potassium nutrition under normal conditions. Under saline conditions, the protein mediates resistance to salt stress through the intracellular compartmentalization of potassium to regulate cellular pH and K(+) homeostasis.

Show MeSH
Related in: MedlinePlus