Isolation and Comparative Analysis of Two Na⁺/H⁺ Antiporter NHX2 Genes from Pyrus betulaefolia

Abstract

The vacuolar Na⁺/H⁺ antiporter NHX gene is a salt tolerance determinant in higher plants. Pyrus betulaefolia, a popular rootstock in Asia, can improve pear salt tolerance through grafting. In this study, two novel NHX genes were isolated from P. betulaefolia using its NaCl-treated transcriptome information. Both PbNHX2.1 and PbNHX2.2 have typical DNA structures and conservative protein motifs often found in vacuolar Na⁺/H⁺ antiporters. They are classified as the same NHXs in the phylogenetic tree. Furthermore, both PbNHX2s localized to the plant cell tonoplast. The NaCl treatment strongly induced the expression of PbNHX2.1 in shoots and PbNHX2.2 in the whole plant, but the transcript levels of PbNHX2.2 were much higher. This result meant that they have diverse functions during sodium ion transport in P. betulaefolia. The PbNHX2.2 transcription was influenced by polyethylene glycol and abscisic acid (ABA). An ABA cis-acting element was found in its promoter region, which indicated that PbNHX2.2 transcriptional regulation under salt and osmotic stresses may be ABA-dependent. Yeast recombined experiments revealed that PbNHX2.1 and PbNHX2.2 restored, with different efficacies, the Na⁺-sensitive phenotype of a endosomal/vacuolar Na⁺/H⁺ antiporter mutant, AXT3. These PbNHX2s proteins, especially PbNHX2.2, facilitated Na⁺ ion transport and maintained intracellular K⁺ status. The results suggest that PbNHX2.2 was a salt tolerance determinant and had a major function in the vacuolar compartmentalization of Na⁺. The information on gene features, transcriptional pattern, and the basic function of the NHX family members in P. betulaefolia will help reveal the molecular mechanism underlying salt tolerance in this species.