In the rice genome, there are at least five NHX members with OsNHX1-OsNHX4 belonging to the type-I and OsNHX5 to the type-II . The operation of the NHXs also affects the pH gradients across the different luminal compartments . Both type-I and type-II NHXs have different subcellular localization; they may have different mechanisms in salt tolerance, and other not yet defined functions. Large efforts have been paid to increasing the plant salt tolerance by over expressing NHX orthologue genes from different species. In rice, over expression of the NHX1 homologue genes from Oryza sativa, Chenopodium glaucum and Atriplex dimorphostegia resulted in the enhanced tolerance to salt stress . However, these NHX proteins actually mediate both Na+ /H+ and K+ /H+ exchange and their functions cannot be solely explained by accumulating Na+ into vacuole . The type-II NHXs have also been shown to be involved in salt tolerance. AtNHX5 and AtNHX6 are the only two type-II NHXs inArabidopsis. Although their function appears to be redundant, the double knockout Atnhx5nhx6 displayed high sensitivity to salt stress . In tomato, the over expression of LeNHX2, a type-II NHX located in prevacuolar compartments and Golgi,industrial drying racks enhanced salt tolerance at high external K+ levels . Plants use a number of strategies to deal with high salinity .
Halophytes rely heavily on the homoeostasis of three major inorganic ions to maintain their osmotic and turgor pressure under saline conditions, while glycophytes predominantly increase the synthesis of compatible solutes . For salt tolerant plants grown in high external saline conditions, the efficient compartmentation of Na+ into vacuole and other cell organs via intracellular NHXs is of importance, together with the extrusion of Na+ at the roots, via plasma membrane-bound antiporters such as SOS . Jerusalem artichoke belonging to the same Asteraceae family as sunflower is an herbaceous perennial plant that have potential as a bio refinery crop . H. tuberosus is highly tolerant to infertile, drought and saline stresses and its variety Nanyu No. 1 could fully emerge in the coastal region containing 1% salt at soil surface layer and grow well in soil containing 0.5%–0.6% salt or under irrigation with 50%–75% sea water . In this study, we isolated two putative NHX genes, HtNHX1 and HtNHX2, from H. tuberosus cv. NY-1 and examined their roles in enhancing the tolerance of salt stress and nutrient deficiency in rice, Arabidopsis and yeast. Our results indicate that HtNHX2 could function in enhancing the plant tolerance to salinity stress and improving plant nutrient efficiency.Considering the essential roles of K+ in balancing uptake and distribution of anions, particularly, nitrate and phosphate and improving plant growth, we characterized the effects of expressing HtNHX1 and HtNHX2 on enhancing the tolerance of rice to nutrient deficiency. The reduction in nutrient supply to 1/4 of its full strength decreased the growth and biomass of WT and HtNHX1-expressing rice equally while did not affect the growth of HtNHX2-expressing rice plants .
The HtNHX2 lines showed about 35%, 25% and 45% increase in total biomass, N and P contents, respectively, in comparison with WT , indicating that HtNHX2 expression could remarkably enhance the root acquisition of N and P, thus improving plant growth under limited nutrient supply conditions. In contrast, N and P contents in the HtNHX1 lines were lower than those in WT. 15N quantification analysis of rice grown in low N and K conditions showed that HtNHX2 expression did not alter root N contents, but significantly increased shoot N contents , suggesting the enhanced N translocation from shoot to root, in addition to the increase in total N uptake. The notion of the role of HtNHX2, but not HtNHX1, in enhancing K+ uptake and mediating N and P accumulation was further supported by the growth of rice plants in infertile paddy fields . Compared to WT, HtNHX1 lines showed 20% lower straw weight and 40% lower grain yield, resulting in significant lower grain harvest index due to a larger portion of unfilled grains . The HtNHX1 lines contained significant higher total N, similar P and lower K+ in the straw at mature stage . Remarkably, the HtNHX2 lines showed 45% increase of total grain yield and 90%, 40% and 13% higher N, P and K+ contents in the straw .Plant NHX-type antiporters play important roles in salt tolerance and the maintenance of cellular K+ and Na+ homoeostasis and the regulation of intracellular pH. In this study, we cloned two highly identical salt-stress up-regulated NHX orthologues genes, HtNHX1 and HtNHX2 from H. tuberosus . HtNHX1 and HtNHX2 share identical 50 -UTR, 30 -UTR and coding sequences, except for a fragment of consecutive 342 bp in HtNHX1 which is absent in HtNHX2. Whether the two genes were alternatively spliced or evolutionary independently generated is not clear. According to their sequences, both genes are predicted to belong to the type-I family and to locate at the vacuole .
The ability of HtNHX1 but not HtNHX2 to confer resistance to hygromycin in yeast and Arabidopsis and different cellular localization in rice would suggest functional differences between the two isoforms. Notably, the role of HtNHX2 in conferring tolerance to nutrient deficiency, in addition to conferring tolerance to salinity, provides a novel insight on NHX functions .We observed that HtNHX1 or HtNHX2 improved rice tolerance to salt stress , but they did not have significant influence on growth and uptake of K+ and Na+ at normal growth conditions . At salinized conditions, HtNHX1 increased rice K+ and Na+ accumulation, that is keeping the same ratio of K+ /Na+ as that in WT . This indicated that HtNHX1 indistinguishably transported K+ and Na+ , which is similar to that of AtNHX1 and AtNHX2 in Arabidopsis . In addition, the K+ and Na+ contents of plant expressing HtNHX1 were higher in the roots and the shoots than in WT plants , supporting the role of HtNHX1 as type-I NHXs in sequestering Na+ into the vacuoles . Interestingly, the accumulation and distribution of K+ and Na+ in plants expressing HtNHX2 differed from that seen in HtNHX1 plants, as HtNHX2 plants accumulated relatively more Na+ and K+ . These results were in agreement with the localization of HtNHX2 to intracellular compartments other than the vacuole . Endosomal trafficking and the vesicle fusion to the vacuole are important components of the response of plant cells to salinity. Transporters playing roles in cation sequestration, such as NHX1 and H+ -pumps ,commercial greenhouse benches depend on vesicular trafficking for their delivery to the tonoplast. Thus, the expression of endosomal NHXs may influence protein trafficking from the Golgi/TGN to the vacuoles , necessary for the response to high salinity. The upregulation of endosomal NHXs, such as AtNHX5 in response to salt stress, but not to osmotic shock, supported the role of endosomal AtNHXs . In addition, salinity induced bulk endocytosis, promoted the rapid increase in vacuolar volume and the accumulation of sodium into the vacuole . These results suggest that the over expression of endosomal NHXs, such as HtNHX2, contributed to salt tolerance through several mechanisms: increasing vesicle fusion to the vacuole, contributing to the accumulation of Na+ and by increasing the trafficking of transporters that could explain the improved nutrient uptake in the transgenic plants. HtNHX1 and HtNHX2 rescued the salt sensitivity of the Arabidopsis Atnhx5 nhx6 double mutant at the same extent , but HtNHX1 was more effective than HtNHX2 in conferring tolerance of Arabidopsis to external hygromycin . It has been shown that AtNHX5 and AtNHX6 located at endosomes, TGN and prevacuolar compartments and nhx5 nhx6 double knockouts displayed abnormal vesicular trafficking and sensitivity to salinity . In rice, HtNHX2 showed similar cellular localization as AtNHX5 ; thus, it is plausible that HtNHX2 may play a similar role as AtNHX5 in the regulation of endosomal ion and pH homoeostasis. Nevertheless, these results should be taken with some caution as both HtNHX1 and HtNHX2 were expressed under the control of a constitutive promoter and abnormal localization due to unregulated expression cannot be ruled out.Potassium is a plant essential nutrient, and vacuolar K+ plays roles in the regulation of cellular volume and tissue expansion. Although plants grown under different K+ supply can change their vacuolar K+ contents, cytosolic K+ is maintained at a relative constant level of around 60–100 mM .
The over expression of AtNHX1 in tomato led to increased vacuolar K+ accumulation and higher tissue K+ contents. In the knockout Atnhx1, Apse et al. reported a reduction in K+ /H+ and Na+ / H+ exchange and reduced cell expansion. Bassil et al. observed lower vacuole pH and K+ concentration in the nhx1 nhx2 mutant, further supporting the role of AtNHX1 and AtNHX2 in driving the uptake of K+ into the vacuole. The expression of HtNHX2 improved plant growth and grain yield at low K+ concentrations , and the tissue K+ contents of the transgenic plants were higher than the WT and HtNHX1-expressing plants. Notably, HtNHX2 localization appeared to be similar to that of AtNHX5 , that is intracellular vesicles, TGN. , thus associated with vesicular trafficking and storage protein sorting . In yeast, nhx1 mutants have been shown to have impaired vacuolar biogenesis and protein sorting . In tomato, LeNHX2 colocalized with prevacuolar and Golgi markers and appeared to be more selective to K+ than Na+ . Interestingly, HtNHX2 lacks a continuous 114 amino acids stretch at predicted six to eight transmembrane domains of HtNHX1 . This stretch also comprises a segment of eight amino acids that is absent in type-II NHXs . Although the occurrence of a shorter HtNHX2isoform retaining its transport function has not been reported before, shorter AtNHX1 isoforms have been reported . Using DNA shuffling mutagenesis, Xu et al. generated a 296 amino acids deleted AtNHX1, AtNHXS1, in which, several transmembrane domains and the C-terminus hydrophilic tail containing the CaM-binding domain were deleted. Interestingly, such large fragment deletion did not alter its vacuolar localization but increased the Na+ /K+ selectivity and Na+ /H+ exchange activity, enhancing the NaCl tolerance of yeast expressing AtNHXS1 . Notably, plants expressing the endosomal HtNHX2 accumulated more K+ and Na+ than the transgenic rice expressing the vacuolar HtNHX1. Although it is possible to speculate that the deletion in HtNHX2 resulted in increased transport activity of the antiporter, a detailed structure/function analysis is needed to assess this point. The increased tolerance to nutrient deficiency displayed by transgenic rice plants expressing HtNHX2 was associated with increased K+ contents and enhanced N assimilation as seen by the increase in 15N translocation from root to shoot and the amounts of N, P and K+ in the straw of plants grown in infertile soil . K+ is the major accompanying ion for the translocation of NO3 and sucrose in plants . About 40%–90% of root acquired K+ could be retranslocated from the shoot via the phloem and recycled through the roots , and K+ recycling in plants can act as an important signal for feedback control of nutrient uptake . It could be speculated that the expression of HtNHX2 improved the synthesis/delivery of the transporters of N, P and K+ to plasma membranes by improving vesicular trafficking and/or protein targeting . The functions of NHX-type transporters have been extensively studied , and the effects of the over expression of NHXs on salt tolerance have been shown in different crop species . Here, we show that although the expression of HtNHX1 conferred salt tolerance, only the expression of HtNHX2, a shorter isoform ot the known type-II NHXs, conferred salt tolerance under nutrient stress conditions.The tubers of H. tuberosus genotype NY-1 after dormancy were germinated on moist sand in an incubator. Uniformly germinated slices of the buds were selected, sown in sands and then transplanted into 1/2 Hoagland nutrient solution in the greenhouse as described previously . After emergence of the fourth leaf, the seedlings were treated with 50, 100, 200 and 300 mM NaCl for 24 h or with 100 mM NaCl for 12 h before the sampling for analysis of HtNHX1 and HtNHX2 expression. Each treatment was replicated three times. Arabidopsis thaliana wild-type and nhx5 nhx6 double knockout mutants were provided by Professor Eduardo Blumwald at University of California .