New isoform of Barley vacuolar (HvNHX3) Na+/H+ - antiporter: expression and immunolocalization
2009
Roslyakova, T. V. | Lasareva, E. M. | Kononenko, N. V. | Babakov, A. V., All-Russia Research and Development Inst. of Agricultural Biotechnology, Moscow (Russian Federation)
In this study we have isolated and identified a gene of the HvNHX3 antiporter from barley plants, which encodes a new isoform of vacuolar Na+/H+ -antiporter. The gene product we have found is expressed both in roots and shoots of barley seedlings. The antiporter has 541 amino acid residues; we were able to estimate the total molecular mass of the protein product (59.7 killo Dalton), which was later confirmed by electrophoresis followed by Western blotting. Amino acid sequence alignment of HvNHX3 with other Na+/H+-antiporters showed the closest related antiporter with homology 95% is HbNHXl isolated from wild type barley Hordeum brevisubulatum, which is growing on salt-marsh (solonchak) soils. We quantitatively measured expression of the HvNHX3 both in barley roots and shoots. The resujts showed a significant increase in the amount of HvNHX3 mRNA under salt stress in both organs. Surprisingly, the amount of the HvNHX3 protein detected by Western blot analysis in roots did not show any difference; however, the amount of the protein in leaves was significantly increased. Immunolocalization of HvNHX3 in roots showed that the protein is ubiquitously present in all root tissues. Control plants showed cluster-staining of the protein, while salt treated plants showed granular distribution. We hypothesize that by changing cluster organization the antiporter reg¬ulates its function. We conclude that activation of the HvNHX3 isoform in roots by cluster dissociation together with increasing amount of the antiporter in leaves under salt stress will facilitate Na+/H+ exchange on vacuolar membrane and, as a result, the Na+ removal from cytoplasm will be enhanced. We presume that this mechanism can explain the increasing resistance of plants to higher salt concentration in their environment
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