| To cope with the detrimental effects of Na+, plants have evolved several strategies such as (i) restricting Na+ influx, (ii) maintaining active Na+ efflux, (iii)compartmentalization of Na+. Plants in saline habitats have acquired mechanisms that allow the selective uptake of K+ in the face of considerable competition from Na+. Some halophytes can accumulate Na+ in its vacuole to adjust to osmotic stress. Ion transport across the tonoplast into vacuoles, is energized by a proton motive force (PMF) generated by the vacuolar H+-ATPase (V-ATPase) and a H+-pyrophosphatase (V-PPiase). Na+ compartmentalization provides a mechanism not only maintaining a low Na+ concentration and keep a high K+ concentration in the cytosol, but also osmotic balance. The overexpression of tonoplast Na+/H+ antiporter genes increased salt tolerance in different plant species, indicating that tonoplast Na+/H+ antiporter (NHX) plays an important role in plant salt stress.Halostachys caspica (Bieb.) C. A. Mey is an extremely salt-tolerant short shrub grown in wilderness or semi-desert salt-alkaloid land in Xinjiang. The morphology of Halostachys caspica has unique character, the stem and leaf have evolved as assimilating shoots. Halostachys caspica can grow normally when exposed to extreme salinity (700 mmol/L NaCl) and the physiological responses of Halostachys caspica have been investigated in our previous work. The study on the molecular mechanism of salt tolerance in Halostachys caspica may provide some clues for improving crops with various traits, and contribute to development of salt tolerant varieties by genetic engineering.According to sequences of vacuolar Na+/H+ antiporter genes from several Xinjiang halophytes, a new vacuolar Na+/H+ antiporter gene (HcNHX1) from the halophyte Halostachys caspica was obtained by RACE and RT-PCR methods with primers corresponding to conserved regions of the coding sequences. The full length of HcNHX1 cDNA obtained was 1,983 bp and contained a 1,656 bp open reading frame sequence which encoded a deduced protein of 551 amino acid residues.Multiple alignment revealed a high degree of homology between the putative amino acid sequences of HcNHX1 and putative vacuolar Na+/H+ antiporter of other higher plants (96~72%). The highest identity was found to be 94% similarity with K. foliatum, a salt-tolerant shrub. The phylogenetic analysis showed that HcNHX1 formed a clade with the most closely related plant NHX homolog, which was distinct from the cluster of plasma membrane Na+/H+ antiporter such as AtSOS1, OsSOS1 and TaSOS1. It also can be seen from the phylogenetic analysis that the HcNHX1 was evolutionally more closely related to dicot halophyte of Chenopodiaceae, such as S. salsa, A. gmelini, C. glaucum, K. foliatum. A hydropathy plot generated by the TMpred program indicated that the HcNHX1 was consisted of 12 putative hydrophobic regions. These results suggested that HcNHX1 is a member of the tonoplast Na+/H+ antiporter gene family.Semi-quantative PCR results showed that the expression level of HcNHX1 gene was enhanced strongly as the NaCl concentration increased, indicating that the expression of HcNHX1 was induced by salt stress. Expression pattern under ABA induction was investigated, and the results revealed that expression of HcNHX1 was induced by ABA, indicating that HcNHX1 may be involved in ABA-dependent pathway.HcNHX1 was over-expressed in Arabidopsis by floral-dip method. PCR and Northern blot analysis of the transgenic plants showed that HcNHX1 gene was successfully expressed in transformed Arabidopsis. Transgenic lines grew more vigorously than the wild type (WT) under salt stress. The analysis of ion contents indicated that under salt stress, the transgenic plants compartmentalized more Na+ in the rosette leaves compared with wild-type plants. Taking together, these results suggest that the product of the novel gene HcNHX1 from halophyte Halostachys caspica is a functional tonoplast Na+/H+ antiporter. Salt tolerance was conferred by over-expression of HcNHX1 in Arabidopsis.
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