Molecular Analysis of the Na+/H+ Exchanger Gene Family and Its Role in Salt Stress in Medicago Truncatula.

High salinity in irrigation water is detrimental to plant growth and productivity. Plants develop various mechanisms and strategies to cope with salinity. One important mechanism is keeping the cytosolic Na⁺ concentration low by sequestering Na⁺ in vacuoles, a process facilitated by Na⁺/H⁺ exchangers (NHX). There are eight NHX genes (NHX1 through NHX8) identified and characterized in Arabidopsis. Bioinformatic analysis of the known Arabidopsis genes using sequence homology, exon-intron structure, domain structure, gene ontology, KEGG annotations, enabled us to identify corresponding Medicago truncatula genes (MtNHX1, MtNHX2 MtNHX3 MtNHX4 MtNHX6 and MtNHX7). There were no orthologs identified for AtNHX5 and AtNHX8. Twelve transmembrane domains and an Amiloride binding site were conserved in five out of six MtNHX proteins. Evolutionary relationships among NHX family members in A. thaliana, Glycine max, Phaseolus vulgaris and M. truncatula revealed that each individual NHX class (Class I:  MtNHX1 through 4; Class II: MtNHX6; Class III: MtNHX7) falls under a separate clade. In salinity stress experiments, M. truncatula exhibited ~20% reduction in biomass. Sodium contents increased by 178% and 75% in leaves and roots, respectively and Cl^- contents increased by 152% and 162%, respectively, in the salinity treatment. Our results revealed that under salt stress, Na⁺ exclusion may be responsible for the relatively smaller increase in Na⁺ concentration as compared to Cl^-. Decline in tissue K⁺ concentration under salinity was not surprising as some antiporters play important role in transporting both Na⁺ and K⁺. Expression analyses revealed that MtNHX1, MtNHX6 and MtNHX7 display high expression in roots and leaves, whereas MtNHX2 and MtNHX3 had low expression. MtNHX3, MtNHX6, MtNHX7 were induced in roots under salinity stress. Expression analyses results indicate that in M. truncatula sequestering Na⁺ into vacuoles may not be the principle component trait of the salt tolerance mechanism and other component traits may be pivotal.