Functional Characterization of a Wheat NHX Antiporter Gene TaNHX2 That Encodes a K+/H+ Exchanger

Yuanyuan Xu,Jianchun Guo,Zhihui Xia,Sha Hong,Xingyu Jiang,Dangqun Cui,Yang Zhou,Haixia Xu,Alvaro Galli

doi:10.1371/journal.pone.0078098

Yuanyuan Xu, Jianchun Guo + Show 7 more

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https://doi.org/10.1371/journal.pone.0078098

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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.

Highlights

Salts present in soils can enter the cytosol of plants, where they are toxic to important physiological and biochemical processes, and inhibit plant growth and development, thereby significantly reducing yield
Multiple alignments of the deduced amino acid sequences of TaNHX2 with two other wheat NHX antiporters, one tomato NHX antiporter and six Arabidopsis NHX antiporters showed that sequence identities between them are 66.24% with TaNHX1 (AAK76737), 69.82% with TaNHX3, 26.51% with LeNHX2 (AJ306631), 70.17% with AtNHX1 (NM_122597), 71.95% with AtNHX2 (NM_111375), 53.86% with AtNHX3 (NM_124929), 64.33% with AtNHX4 (NM_111512), 27.62% with AtNHX5 (NM_104315) and 27.17% with AtNHX6 (NM_106609) using DNAMAN 6.0 software (Fig. 1)
Phylogenetic analysis showed that the three wheat NHX antiporters were closer to Arabidopsis AtNHX1-4 which localize in the tonoplast [11,12,31,32] compared with Arabidopsis AtNHX5-6 and tomato LeNHX2, which are endomembrane-bound proteins [16,17]

Summary

Introduction

Salts (mainly NaCl) present in soils can enter the cytosol of plants, where they are toxic to important physiological and biochemical processes, and inhibit plant growth and development, thereby significantly reducing yield. No toxic substance restricts plant growth more than salts; salt stress presents an increasing threat to plant agriculture [1]. To tolerate high levels of Na+ ions, plants cells must be able to transport Na+ out of the cytoplasm to the external medium or sequester it to the vacuole, maintaining the cytosolic Na+ concentration at a non-toxic level. Sodium transport into vacuoles can be accomplished by the operation of tonoplastbound NHX proteins, which function as Na+/H+ antiporters, driven by the electrochemical gradient of protons generated by the vacuolar H+-ATPase and H+-PPase. NHX antiporters have important roles in the plant’s response to salt stress [6]

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