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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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Enhanced salt tolerance of transgenic yeast cells expressing TaNHX2.Yeast cells of recombinant strains, mutant and wild-type were grown to saturation in APG medium. Ten microliters of serial decimal dilutions were spotted onto plates of APG medium supplemented with 0 (control), 30 or 60 mM NaCl (A) and 0.25 or 0.5 M KCl (B) as indicated. Plates were incubated at 30°C for 3-5d. TaNHX2: mutant strain AXT3K transformed with plasmid pYES2-TaNHX2; pYES2: mutant strain AXT3K transformed with the empty plasmid pYES2; W303: wild-type strain W303.
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pone-0078098-g005: Enhanced salt tolerance of transgenic yeast cells expressing TaNHX2.Yeast cells of recombinant strains, mutant and wild-type were grown to saturation in APG medium. Ten microliters of serial decimal dilutions were spotted onto plates of APG medium supplemented with 0 (control), 30 or 60 mM NaCl (A) and 0.25 or 0.5 M KCl (B) as indicated. Plates were incubated at 30°C for 3-5d. TaNHX2: mutant strain AXT3K transformed with plasmid pYES2-TaNHX2; pYES2: mutant strain AXT3K transformed with the empty plasmid pYES2; W303: wild-type strain W303.

Mentions: The recombinant plasmid pYES2–TaNHX2-His was obtained by inserting the TaNHX2-His fragment into pYES2 between the GAL1 promoter and the CYC1 terminator. The plasmid pYES2–TaNHX2-His was then transferred into the yeast strain AXT3K, in which the major endogenous sodium transporter are disrupted, to test the function of TaNHX2. AXT3K transformed with empty vector pYES2 was sensitive to 30 mM and 60 mM NaCl (Figure 5A) as well as 0.25 M and 0.5 M KCl (Figure 5B). The responses of mutant strain AXT3K and AXT3K transformed with empty vector pYES2 to NaCl and KCl stresses were similar (data not shown). TaNHX2 expression could complement the sensitivity of AXT3K to NaCl and KCl stresses.


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)

Enhanced salt tolerance of transgenic yeast cells expressing TaNHX2.Yeast cells of recombinant strains, mutant and wild-type were grown to saturation in APG medium. Ten microliters of serial decimal dilutions were spotted onto plates of APG medium supplemented with 0 (control), 30 or 60 mM NaCl (A) and 0.25 or 0.5 M KCl (B) as indicated. Plates were incubated at 30°C for 3-5d. TaNHX2: mutant strain AXT3K transformed with plasmid pYES2-TaNHX2; pYES2: mutant strain AXT3K transformed with the empty plasmid pYES2; W303: wild-type strain W303.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0078098-g005: Enhanced salt tolerance of transgenic yeast cells expressing TaNHX2.Yeast cells of recombinant strains, mutant and wild-type were grown to saturation in APG medium. Ten microliters of serial decimal dilutions were spotted onto plates of APG medium supplemented with 0 (control), 30 or 60 mM NaCl (A) and 0.25 or 0.5 M KCl (B) as indicated. Plates were incubated at 30°C for 3-5d. TaNHX2: mutant strain AXT3K transformed with plasmid pYES2-TaNHX2; pYES2: mutant strain AXT3K transformed with the empty plasmid pYES2; W303: wild-type strain W303.
Mentions: The recombinant plasmid pYES2–TaNHX2-His was obtained by inserting the TaNHX2-His fragment into pYES2 between the GAL1 promoter and the CYC1 terminator. The plasmid pYES2–TaNHX2-His was then transferred into the yeast strain AXT3K, in which the major endogenous sodium transporter are disrupted, to test the function of TaNHX2. AXT3K transformed with empty vector pYES2 was sensitive to 30 mM and 60 mM NaCl (Figure 5A) as well as 0.25 M and 0.5 M KCl (Figure 5B). The responses of mutant strain AXT3K and AXT3K transformed with empty vector pYES2 to NaCl and KCl stresses were similar (data not shown). TaNHX2 expression could complement the sensitivity of AXT3K to NaCl and KCl stresses.

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