- Na+/H+antiporters catalyze the exchange of Na+ for H+ across membranes; and they are responsible for regulating internal pH, cell volume, and the sodium level in the cytoplasm. Antiporters are found in animals, yeast, bacteria, and plants and have been reported to be localized in the plasma membrane and in various organelles including the prevacuolar compartment, vacuolar membranes, mid- to trans-Golgi network, trans-Golgi network, early recycling endosomes (HsNHE6), late recycling endosomes (HsNHE9), and in chloroplasts. It has been suggested that plant cells do not have Na+/K+ -ATPase or Na+ - ATPase, which acts to exclude Na + from animal and fungal cells. Therefore, in plants, H+ -ATPase and H+ -inorganic pyrophosphatase are the primary active transporters that generate an H + -motive force, whereas the Na+ /H+ antiporter is the main transporter of Na+ .
- Control of intracellular ion concentrations is essential for ion homeostasis. In plants under salt stress, Na+ can enter the cells via several pathways and can become toxic to cytosolic enzymes at high concentrations. Therefore, plant cells must maintain a high concentration of K+ and a low concentration of Na+ in the cytosol and must extrude excessive Na+ or compartmentalize it into vacuoles. Na+ efflux is catalyzed by a plasma membrane Na+/H+ antiporter that is encoded by the AtSOS1 gene in A. thaliana. By contrast, a vacuolar Na+/H+ antiporter catalyzes the sequestration of Na+in vacuoles. The sequestration lowers the Na + concentration in the cytoplasm and also contributes to osmotic adjustment to maintain water uptake from saline solutions, which suggests that compartmentalizing Na + into vacuoles is an essential strategy for salt tolerance in plants.
- The genomic organization of OsNHX5 is graphically presented in Figure 1. OsNHX5 had 22 exons
- OsNHX expression was also enhanced under submergence, and the levels of OsNHX1 and OsNHX5 transcripts agreed well with those of seedling vigor under submergence.
- OsNHX1, OsNHX2, OsNHX3, and OsNHX5 can suppress the Na+, Li+, and hygromycin sensitivity of yeast nhx1 mutants and their sensitivity to a high K+ concentration. The expression of OsNHX1, OsNHX2, OsNHX3, and OsNHX5 is regulated differently in rice tissues and is increased by salt stress, hyperosmotic stress, and ABA.
- OsNHX1 and OsNHX5 play different roles in these rice tissues, although the antiporters cooperate in most tissues.
1. Under the same experimental conditions as the ones used for the ethylene biosynthesis genes, transcripts of OsNHX2 and OsNHX3 could not be detected. For three other paralogs, OsNHX1, 4 and 5, Hien Thi Thu Vu et al. observed marked induction under the submergence stress. The level of induction was higher in the vigorous cultivars Nipponbare and FR13A than in non-vigorous Kasalath and IR42. The amount of OsNHX3 and OsNHX5 transcripts under submergence was lowest in the most non-vigorous IR42.
2. Hien Thi Thu Vu et al. therefore studied the expression profiles of Na+/H+ exchanger gene OsNHX using submerged rice seedlings. It showed that three paralogs of OsNHX responded positively to the submergence stress and that the level of enhancement appeared to agree with the level of seedling vigor among the four cultivars studied. Although variation in gene expression itself could not be taken as a valid indicator of mechanistic evidence, this observation indicates the necessity of further study of the role of OsNHX gene expression in seedling vigor under submergence in rice.
3. Fukuda et al. examined the expression of OsNHX1, OsNHX2,OsNHX3, and 5 in various rice tissues by Northern-blot analysis with total RNA extracted from 8-day-old rice seedlings and 12-weekold rice (7 days after heading). Expression of these genes was regulated differently in different rice tissues (Fig. 2). Compared with other tissues, and those of OsNHX5 were higher in panicles, flag leaf sheaths, seedling roots, and flag leaf blades.
4. OsNHX5 promoter–GUS expression pattern in transgenic rice plants:
- OsNHX5 promoter–GUS expression was observed in the stele of roots, the emerging part of lateral roots, the shoot vascular bundle, water pore, and the basal part of shoots where OsNHX1 promoter activity was also higher (Fig. 3a–g).
- GUS activity was also detected in the vascular bundles, especially the companion cells and xylem parenchyma cells, of culms and flag leaf blades and sheaths of mature plants in the heading stage and the nerves of both the palea and lemma, as for OsNHX1 promoter–GUS
activity (Fig. 3h–q).
- However, stronger OsNHX5 promoter activity was detected in the root tip, especially the root cap and epidermis cells, and pollen grains compared with the OsNHX1 promoter (Fig. 3b, p–r). These findings suggest that OsNHX1 and OsNHX5 play different roles in these rice tissues, although the antiporters cooperate in most tissues.
5. The expression of OsNHX1, 2, 3, and 5 is regulated differently in different rice tissues. The transcript levels of all genes were higher in flag leaf sheaths, and those of OsNHX1, 2, and 5 were higher in panicles .
- The OsNHX proteins shared between 29 and 75% identity. OsNHX1 through 4 in particular shared high similarity, with more than 70% identity among OsNHX1 through 3 and more than 50% identity among OsNHX1 through 4. The sequence of LFFIYLLPPI, which is identified as the binding site of amiloride, an inhibitor of eukaryotic Na+/H+ antiporters, was identical among OsNHX1 through 3 and highly conserved in OsNHX4 and 5. The membrane-spanning segments M5 and M6 of OsNHX1, which are well conserved in the eukaryotic Na+/H+ antiporters, also shared high similarity with OsNHX2 through 5, and all of the OsNHX proteins contained the residues important for Na+/H+ antiport activity.
- OsNHX1 through 4, AtNHX1 through 4, and other most plant NHXtype antiporters are classified into a type I group, and OsNHX5, AtNHX5, AtNHX6, and LeNHX2 from L. esculentum are classified into the type II group (Fig. 4).
- Arabidopsis contains six NHX genes and rice has five, which are distributed in a similar way: AtNHX1–4 and OsNHX1–4 constitute class
I, and AtNHX5–6 and OsNHX5 constitute class II (Fig. 5). Members of the class-I category of Arabidopsis and rice show 54–87% similarity. Similarity among class-II members is 72–79%, but they are only 21–23% similar to class-I isoforms. These data indicate that divergence between class-I and class-II exchangers in plants occurred before the separation of dicotyledons and monocotyledons.
- Pires et al. observe that multiple independent duplication events have occurred throughout the evolutionary history of the NHX family. Based on the reconciled phylogeny, Pires et al. estimate 27 independent gene duplication and 40 gene loss events during the diversification of this gene family.
- The observed mode of OsACS, OsACO and OsNHX expression under submergence suggests that these genes can be potential targets for understanding the mechanism regulating seedling vigor under submergence at the post-germination stage in rice.
- The Na+/H+exchanger that catalyzes the exchange of Na+for H+across membranes, contributes to regulation of internal pH, cell volume, and sodium level in the cytoplasm.
- Na+/H+ antiporters, which catalyze the exchange of Na+ for H+ across membranes, contribute to the regulation of internal pH, cell volume and the sodium level in the cytoplasm. The antiporters are widespread membrane proteins found in animals, yeasts, bacteria and plants. In particular, vacuolar Na+/H+ antiporters, which compartmentalize Na+ into the vacuoles for detoxification, have been investigated as the key to salt tolerance in yeasts and plants.
- Amino acid sequence similarities together with the phylogenetic analysis and the deduced exon–intron structures indicated that the OsNHX family antiporters form two distinct subgroups: OsNHX1 through 4 and OsNHX5. In A. thaliana, Yokoi et al. (2002) categorized the AtNHX family antiporters into the subgroups AtNHX1 through 4 and AtNHX5 and 6. Therefore, OsNHX1 through 4, AtNHX1 through 4, and other most plant NHX-type antiporters are classified into a type I group, and OsNHX5, AtNHX5, AtNHX6, and LeNHX2 from L. esculentum are classified into the type II group. Pardo et al. (2006) also classified the antiporters into the same groups. LeNHX2 catalyzes K+/H+ exchange, but scarcely catalyzes Na+/H+ exchange, and co-localizes with prevacuolar and Golgi markers in plants. These results suggest that the ionic selectivities and physiological roles of type II NHX-type antiporters likely differ from those of type I NHX-type antiporters.
Labs working on this gene
- Division of Plant Sciences, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan
- Graduate School of Life and Environmental Sciences, Tsukuba University, Tennoudai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan
- Kobe University, Graduate School of Agricultural Science, Kobe, Japan
- Agricultural Genetics Institute (AGI), Department of Molecular Biology, Hanoi, Vietnam
- Philippine Rice Research Institute (PhilRice), Plant Breeding and Biotechnology Division, Science City of Munoz, Philippines
- Hyogo Institute of Agriculture, Forestry and Fishery, Kasai, Japan
- Instituto de Recursos Naturales y Agrobiologı´a, Consejo Superior de Investigaciones Cientı´ficas, Reina Mercedes 10, Sevilla 41012, Spain
- Fukuda A, Nakamura A, Hara N, et al. Molecular and functional analyses of rice NHX-type Na+/H+ antiporter genes[J]. Planta, 2011, 233(1): 175-188.
- Vu H T T, Manangkil O E, Mori N, et al. Induction and Repression of Gene Expression Mediating Ethylene Biosynthesis and Sodium/Proton Exchange in Rice Seedlings Under Submergence Stress[J]. Biotechnology & Biotechnological Equipment, 2012, 26(3): 2945-2951.
- Pardo J M, Cubero B, Leidi E O, et al. Alkali cation exchangers: roles in cellular homeostasis and stress tolerance[J]. Journal of Experimental Botany, 2006, 57(5): 1181-1199.
- Bowers K, Levi B P, Patel F I, et al. The sodium/proton exchanger Nhx1p is required for endosomal protein trafficking in the yeast Saccharomyces cerevisiae[J]. Molecular Biology of the Cell, 2000, 11(12): 4277-4294.
- Hamada A, Hibino T, Nakamura T, et al. Na+/H+ Antiporter fromSynechocystis Species PCC 6803, Homologous to SOS1, Contains an Aspartic Residue and Long C-Terminal Tail Important for the Carrier Activity[J]. Plant physiology, 2001, 125(1): 437-446.
- Pires I S, Negrão S, Pentony M M, et al. Different evolutionary histories of two cation/proton exchanger gene families in plants[J]. BMC plant biology, 2013, 13(1): 97.
- Orlowski J, Grinstein S. Na+/H+ exchangers of mammalian cells[J]. Journal of Biological Chemistry, 1997, 272(36): 22373-22376.
- Aronson P S. Kinetic properties of the plasma membrane Na+-H+ exchanger[J]. Annual Review of Physiology, 1985, 47(1): 545-560.
- Numata M, Orlowski J. Molecular cloning and characterization of a novel (Na+, K+)/H+ exchanger localized to the trans-Golgi network[J]. Journal of Biological Chemistry, 2001, 276(20): 17387-17394.
- Blumwald E, Aharon G S, Apse M P. Sodium transport in plant cells[J]. Biochimica et Biophysica Acta (BBA)-Biomembranes, 2000, 1465(1): 140-151.