NHX Genes Research Articles

Molecular characterization and expression analysis of the mulberry Na+/H+ exchanger gene family

Na+/H+ exchangers (NHXs) have important roles in cellular pH, and Na+ and K+ homeostasis in plants. Mulberry is not only an important traditional economic woody plant known for its leaves, which are the exclusive food source of the silkworm Bombyx mori, but it can also adapt to many different adverse conditions, including saline environments. However, little is known about the NHXs in this important perennial tree. In this study, we identified and cloned seven putative NHX gene family members from Morus atropurpurea based on a genome-wide analysis of the Morus genome database. A phylogenetic analysis and genomic organization of mulberry NHXs suggested that the mulberry NHX family forms three distinct subgroups. Transcriptome data and real-time PCR of different mulberry varieties under normal culture conditions revealed that the mulberry NHX family has a different tissue-specific pattern in the two mulberry species. The MaNHX genes' expression analyses under different stresses (salt and drought) and signal molecules (abscisic acid, salicylic acid, hydrogen peroxide and methyl jasmonate) revealed that MaNHXs not only could be induced by salt, drought and abscisic acid as describe in the literature, but were also induced by other signal molecules, which indicated that MaNHX members exhibited diverse and complicated expression patterns in different mulberry tissues under various abiotic stresses, phytohormones and plant signaling molecules. Our results provide some insights into new and emerging cellular and physiological functions of this group of H+-coupled cation exchangers, beyond their function in salt tolerance, and also provide the basis for further characterizations of MaNHXs' physiological functions.

Expression of the Vacuolar Na+/H+ Antiporter Gene (NHX1) in Three Plantago Species Differing in Salt Tolerance

We have isolated full-length cDNA clones of the NHX1 gene - encoding the vacuolar Na + /H + antiporter - from three Plantago species ( P. crassifolia , P. coronopus and P. major ) differing in salt tolerance. A phylogenetic tree of their deduced protein sequences and those of NHX1 antiporters previously isolated from other species suggested the presence of some structural features characteristic of the proteins of salt tolerant taxa. The expression patterns of the NHX1 genes in the presence of salt correlated with the relative degree of salt tolerance of the three species: no induction was detected in the least tolerant P. major - and in the glycophyte A. thaliana - and the highest and quickest induction was observed in the most tolerant P. crassifolia . These data support the hypothesis that the NHX1 antiporters play a functional role in the mechanisms of salt tolerance in plants

Assessment of the vacuolar Na+/H+ antiporter (NHX1) transcriptional changes in Leptochloa fusca L. in response to salt and cadmium stresses.

Sodium/proton exchangers (NHX) are key players in plant responses to salinity and have a central role in establishing ion homeostasis. NHXs can be localized in tonoplast or plasma membranes, where they exchange sodium ions for protons, resulting in the removal of ions from the cytosol into vacuole or extracellular spaces. In the present study, the expression pattern of the gene encoding Na+/H+ antiporter in the vacuolarmembrane (NHX1 gene) in Leptochloa fusca (Kallar grass) was measured by a semi- quantitative RT-PCR method under different treatments of NaCl and CdCl2. Results indicated that NaCl positively affected expression levels of LfNHX1, and that the amount of LfNHX1 mRNA increased in conjunction with the rise of salinity pressure, This finding suggests that vacuolar Na+/H+ antiporter might play an important role in the salt tolerance ability of kallar grass. The results also showed that cadmium exposure significantly modulated the mRNA expression of the LfNHX1 gene, suggesting that cadmium exposure disturbed Na+ homeostasis across the tonoplast and decreased the salt tolerance ability of kallar grass.

Differential transcript abundance of salt overly sensitive (SOS) pathway genes is a determinant of salinity stress tolerance of wheat

Salt overly sensitive (SOS) pathway genes, SOS1 (plasma membrane Na+/H+ antiporter), SOS2 (CBL interacting protein kinase 24), and SOS3 (calcineurin B like protein 4) are associated with active efflux of toxic sodium ions (Na+) from cytosol and thus confer salinity tolerance in glycophytic plants such as Arabidopsis. The role of SOS pathway genes SOS2 and SOS3 in salinity tolerance of wheat is rarely studied. One-month-old seedlings of three bread wheat genotypes namely, HD 2009, HD2687 and Kharchia 65 were imposed with two levels of salinity stress (100 and 200 mM NaCl) for 30 days duration. Based on the physiological parameters, genotype Kharchia 65 was highly tolerant, HD 2009 was moderately tolerant and HD 2687 was sensitive to salinity stress. Tolerant genotypes accumulated lesser amount of Na+ in root, stem and leaf tissues. Transcript abundance of SOS1, SOS2 and SOS3 genes was significantly higher in salt tolerant genotypes under long-term salinity and correlated with improved sodium exclusion, and higher potassium/sodium (K+/Na+) ratio. Expression levels of genes involved in vacuolar partitioning of Na+, NHX1 (vacuolar Na+/H+ antiporter) and VP1 (Vacuolar pyrophosphatase) were also higher in salt tolerant wheat genotypes under 200 mM NaCl stress. Partial coding sequences of SOS1, SOS2, SOS3, NHX1 and VP1 genes were cloned and sequenced from the above mentioned three wheat genotypes. The results in the present study demonstrated that SOS pathway of ion homeostasis under salinity stress is conserved across species.

Whole-genome identification and expression analysis of K+ efflux antiporter (KEA) and Na+/H+ antiporter (NHX) families under abiotic stress in soybean

Sodium toxicity and potassium insufficient are important factors affecting the growth and development of soybean in saline soil. As the capacity of plants to maintain a high cytosolic, K+/Na+ ratio is the key determinant of tolerance under salt stress. The aims of the present study were to identify and analyse expression patterns of the soybean K+ efflux antiporter (KEA) gene and Na+/H+ antiporter (NHX) gene family, and to explore their roles under abiotic stress. As a result, 12 soybean GmKEAs genes and 10 soybean GmNHXs genes were identified and analyzed from soybean genome. Interestingly, the novel soybean KEA gene Glyma16g32821 which encodes 11 transmembrane domains were extremely up-regulated and remained high level until 48 h in root after the excessive potassium treatment and lack of potassium treatment, respectively. The novel soybean NHX gene Glyma09g02130 which encodes 10 transmembrane domains were extremely up-regulated and remained high level until 48 h in root with NaCl stress. Imaging of subcellular locations of the two new Glyma16g32821-GFP and Glyma09g02130-GFP fusion proteins indicated all plasma membrane localizations of the two novel soybean genes. The 3D structures indicated that the two soybean novel proteins Glyma09g02130 (NHX) and Glyma16g32821 (KEA) all belong to the cation/hydrogen antiporter family.

A stress responsive gene of Fortunella crassifolia FcSISP functions in salt stress resistance

Exploration of genes functioning in salt tolerance is crucial for generating transgenic plants with enhanced salt tolerance. In this study, we report the isolation and functional characterization of a stress-responsive gene FcSISP from Meiwa kumquat (Fortunella crassifolia). FcSISP encodes a putative protein of 47 amino acids, with a calculated molecular mass of 4.94 kDa and theoretical isoelectric point of 3.76, and was localized in the nucleus. Transcript levels of FcSISP were induced by dehydration, cold, salt and bacterium causing citrus canker, and hormones (salicylic acid and abscisic acid), with the greatest induction under salt treatment. Overexpression of FcSISP in tobacco (Nicotiana nudicaulis) conferred enhanced salt tolerance. The transgenic lines accumulated lower Na+ contents, leading to reduced Na/K ratio, but accumulated more proline than the wild type (WT). Steady state mRNA levels of genes involved in Na+ exchange (three SOS genes and three NHX genes) and proline synthesis (P5CS and P5CR) were higher in the transgenic lines in comparison with WT. Moreover, overexpression of FcSISP in trifoliate orange [Poncirus trifoliata (L.) Raf.], a widely-used and salt-sensitive citrus rootstock, led to elevated salt tolerance. Taken together, the data demonstrate that FcSISP plays a positive role in salt tolerance and that it holds a great potential for engineering salt tolerance in crops.

Overexpression of StNHX1, a Novel Vacuolar Na+/H+ antiporter gene from Solanum torvum, enhances salt tolerance in transgenic vegetable soybean

Soil salinity is the primary limiting factor affecting the growth and production of vegetable soybean [Glycine max (L.) Merrill] worldwide. Plants can survive in salinity conditions through the mechanism of sodium ion (Na+) compartmentalization, during which the vacuolar Na+/H+ antiporter (NHX) genes play a critical role. In the present study, a novel vacuolar NHX gene from Solanum torvum Swartz ‘Torvum Vigor’ (StNHX1; GenBank accession number: JN606860.1) was isolated and transferred into the vegetable soybean ‘Xinliaoxian’ through an Agrobacterium-mediated protocol. The PCR and southern blot analyses confirmed the successful integration of the exogenous genes. The GUS assay and semi-quantitative RT-PCR analysis showed that the alien genes were inherited by the progenies and that the StNHX1 gene was overexpressed in the T3 generation. The overexpression of this gene conferred high tolerance to salt stress. Under 100 mM NaCl conditions, the scorch scores and the Na+ and malondialdehyde (MDA) contents of the leaves of the transgenic lines were significantly lower as compared with the wild-type (WT) plants, while the K+/Na+ ratio, the contents of K+, relative chlorophyll and relative water, and some morphological traits of the transgenic plants were significantly higher than those of the WT. These results indicate that the overexpression of StNHX1 enhances the salt tolerance of vegetable soybean and that StNHX1 is one of the promising target genes that can be manipulated to improve the salinity tolerance of crops.

Quantitative gene expression analysis of some sodium ion transporters under salinity stress in Aeluropus littoralis

Plant sodium transporters activity is one of the most important salt tolerance mechanisms to keep normal status of cytosolic sodium content. In the present study, expression pattern of genes encoding Na+/H+ antiporters in the plasma membrane (SOS1 gene), vacuolar membrane (NHX1 gene) and H+-ATPase pump (VHA gene) in Aeluropus littoralis under different treatments of NaCl was measured by the semi-quantitative RT-PCR method. Our results indicated that root and shoot sodium contents were increased along with increasing salinity pressure. In response to 200 and 400mM NaCl, mRNA level of SOS1 and NHX1 was increased in the shoot and root tissues, while VHA transcripts were increased only under 400mM of NaCl. Transcripts of VHA-c and NHX1 in root were higher than shoot in all treatments. In general, our results indicated that transcriptional level of SOS1, and NHX1 genes induced in parallel with VHA expression may be involved in controlling cytosolic Na+ concentration in A. littoralis.

Different evolutionary histories of two cation/proton exchanger gene families in plants

BackgroundGene duplication events have been proposed to be involved in the adaptation of plants to stress conditions; precisely how is unclear. To address this question, we studied the evolution of two families of antiporters. Cation/proton exchangers are important for normal cell function and in plants, Na+,K+/H+ antiporters have also been implicated in salt tolerance. Two well-known plant cation/proton antiporters are NHX1 and SOS1, which perform Na+ and K+ compartmentalization into the vacuole and Na+ efflux from the cell, respectively. However, our knowledge about the evolution of NHX and SOS1 stress responsive gene families is still limited.ResultsIn this study we performed a comprehensive molecular evolutionary analysis of the NHX and SOS1 families. Using available sequences from a total of 33 plant species, we estimated gene family phylogenies and gene duplication histories, as well as examined heterogeneous selection pressure on amino acid sites. Our results show that, while the NHX family expanded and specialized, the SOS1 family remained a low copy gene family that appears to have undergone neofunctionalization during its evolutionary history. Additionally, we found that both families are under purifying selection although SOS1 is less constrained.ConclusionsWe propose that the different evolution histories are related with the proteins’ function and localization, and that the NHX and SOS1 families are examples of two different evolutionary paths through which duplication events may result in adaptive evolution of stress tolerance.

A newly isolated Na+/H+ antiporter gene, DmNHX1, confers salt tolerance when expressed transiently in Nicotiana benthamiana or stably in Arabidopsis thaliana

Sodium/hydrogen transporters transfer ions across membranes and thus play an important role in pH and electrolyte homeostasis. To further understand the mechanism and function of the plant vacuolar Na+/H+, a new Na+/H+ antiporter, named DmNHX1, was isolated from chrysanthemum (Dendranthema morifolium) and characterized. The total length of DmNHX1 is 1,897 bp, with an open reading frame of 1,653 bp. Functional complementation studies in Saccharomyces cerevisiae strains with mutated endogenous NHX genes showed that the encoded protein, DmNHX1, has a broad cation specificity (Na+, K+, Li+) and confered hygromycin B tolerance. Quantitative RT-PCR revealed that the transcription level of DmNHX1 was induced by salt stress in all studied tissues, especially expressed highest in the leaves. Agrobacterium-mediated transient expression of DmNHX1 in Nicotiana benthamiana enhanced the salt tolerance of leaf discs, as indicated by reduced bleaching and increased chlorophyll content when compared to that of controls. In addition, overexpressing DmNHX1 resulted in enhanced salinity tolerance in transgenic Arabidopsis thaliana. Our studies suggest that DmNHX1 plays the crucial role in salt transport of chrysanthemum and could be applied for the improvement of salt tolerance.

Ion Exchangers NHX1 and NHX2 Mediate Active Potassium Uptake into Vacuoles to Regulate Cell Turgor and Stomatal Function in Arabidopsis

Intracellular NHX proteins are Na(+),K(+)/H(+) antiporters involved in K(+) homeostasis, endosomal pH regulation, and salt tolerance. Proteins NHX1 and NHX2 are the two major tonoplast-localized NHX isoforms. Here, we show that NHX1 and NHX2 have similar expression patterns and identical biochemical activity, and together they account for a significant amount of the Na(+),K(+)/H(+) antiport activity in tonoplast vesicles. Reverse genetics showed functional redundancy of NHX1 and NHX2 genes. Growth of the double mutant nhx1 nhx2 was severely impaired, and plants were extremely sensitive to external K(+). By contrast, nhx1 nhx2 mutants showed similar sensitivity to salinity stress and even greater rates of Na(+) sequestration than the wild type. Double mutants had reduced ability to create the vacuolar K(+) pool, which in turn provoked greater K(+) retention in the cytosol, impaired osmoregulation, and compromised turgor generation for cell expansion. Genes NHX1 and NHX2 were highly expressed in guard cells, and stomatal function was defective in mutant plants, further compromising their ability to regulate water relations. Together, these results show that tonoplast-localized NHX proteins are essential for active K(+) uptake at the tonoplast, for turgor regulation, and for stomatal function.

Plant and Yeast NHX Antiporters: Roles in Membrane TraffickingF

The plant NHX gene family encodes Na(+)/H(+) antiporters which are crucial for salt tolerance, potassium homeostasis and cellular pH regulation. Understanding the role of NHX antiporters in membrane trafficking is becoming an increasingly interesting subject of study. Membrane trafficking is a central cellular process during which proteins, lipids and polysaccharides are continuously exchanged among membrane compartments. Yeast ScNhx1p, a prevacuole/ vacuolar Na(+)/H(+) antiporter, plays an important role in regulating pH to control trafficking out of the endosome. Evidence begins to accumulate that plant NHX antiporters might function in regulating membrane trafficking in plants.

Endosomal Na+ (K+)/H+ Exchanger Nhx1/Vps44 Functions Independently and Downstream of Multivesicular Body Formation

The multivesicular body (MVB) is an endosomal intermediate containing intralumenal vesicles destined for membrane protein degradation in the lysosome. In Saccharomyces cerevisiae, the MVB pathway is composed of 17 evolutionarily conserved ESCRT (endosomal sorting complex required for transport) genes grouped by their vacuole protein sorting Class E mutant phenotypes. Only one integral membrane protein, the endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44, has been assigned to this class, but its role in the MVB pathway has not been directly tested. Herein, we first evaluated the link between Nhx1 and the ESCRT proteins and then used an unbiased phenomics approach to probe the cellular role of Nhx1. Select ESCRT mutants (vps36Δ, vps20Δ, snf7Δ, and bro1Δ) with defects in cargo packaging and intralumenal vesicle formation shared multiple growth phenotypes with nhx1Δ. However, analysis of cellular trafficking and ultrastructural examination by electron microscopy revealed that nhx1Δ cells retain the ability to sort cargo into intralumenal vesicles. In addition, we excluded a role for Nhx1 in Snf7/Bro1-mediated cargo deubiquitylation and Rim101 response to pH stress. Genetic epistasis experiments provided evidence that NHX1 and ESCRT genes function in parallel. A genome-wide screen for single gene deletion mutants that phenocopy nhx1Δ yielded a limited gene set enriched for endosome fusion function, including Rab signaling and actin cytoskeleton reorganization. In light of these findings and the absence of the so-called Class E compartment in nhx1Δ, we eliminated a requirement for Nhx1 in MVB formation and suggest an alternative post-ESCRT role in endosomal membrane fusion.

Expression of LeNHX isoforms in response to salt stress in salt sensitive and salt tolerant tomato species

In general, wild tomato species are more salt tolerant than cultivated species, a trait that is related to enhanced Na(+) accumulation in aerial parts in the wild species, but the molecular basis for these differences is not known. Plant NHX proteins have been suggested to be important for salt tolerance by promoting accumulation of Na(+) or K(+) inside vacuoles. Therefore, differences in expression or activity of NHX proteins in tomato could be at the basis of the enhanced salt tolerance in wild tomato species. To test this hypothesis, we studied the expression level of four NHX genes in the salt sensitive cultivated species Solanum lycopersicum L. cv. Volgogradskij and the salt tolerant wild species Solanum pimpinelifolium L in response to salt stress. First, we determined that in the absence of salt stress, the RNA abundance of LeNHX2, 3 and 4 was comparable in both species, while more LeNHX1 RNA was detected in the tolerant species. LeNHX2 and LeNHX3 showed comparable expression levels and were present in all tissues, while LeNHX4 was expressed above all in stem and fruit tissues. Next, we confirmed that the wild species was more tolerant and accumulated more Na(+) in aerial parts of the plant. This correlated with the observation that salt stress induced especially the LeNHX3 and LeNHX4 isoforms in the tolerant species. These results support a role of NHX genes as determinants of salt tolerance in tomato, inducing enhanced Na(+) accumulation observed in the wild species when grown in the presence of NaCl.

Overexpression of AtNHX5 improves tolerance to both salt and water stress in rice (Oryza sativa L.)

Overexpression of NHX genes has been previously shown to improve salt tolerance of transgenic plants. In this study, transgenic rice plants overexpressing AtNHX5 showed not only high salt tolerance, but also high drought tolerance. Measurements of ion levels indicated that Na+ and K+ contents were all higher in AtNHX5 overexpressing shoots than in wild type (WT) shoots in high saline conditions. After exposure to water deficiency and salt stress, the WT plants all died, while the AtNHX5 overexpressing rice plants had a higher survival rate, dry weight, leaf water content, and leaf chlorophyll contents, accumulated more proline, and had less membrane damage than the WT plants. In addition, seeds of both transgenic and WT plants germinated on 1/2 MS medium supplemented with 250 mM mannitol, but overexpression of AtNHX5 improved the shoot growth of the seedlings. Taken together, the results indicate that AtNHX5 gene could enhance the tolerance of rice plants to multiple environmental stresses by promoting the accumulation of more effective osmolytes (ions or proline) to counter the osmotic stress caused by abiotic factors.

Cloning and characterization of the Salicornia brachiata Na+/H+ antiporter gene SbNHX1 and its expression by abiotic stress

Salinity causes multifarious adverse effects to plants. Plants response to salt stress involves numerous processes that function in coordination to alleviate both cellular hyperosmolarity and ion disequilibrium. A Na(+)/H(+) antiporter NHX1 gene has been isolated from a halophytic plant Salicornia brachiata in this study. Predicted amino acid sequence similarity, protein topology and the presence of functional domains conserved in SbNHX1 classify it as a plant vacuolar NHX gene. The SbNHX1 cDNA has an open reading frame of 1,683 bp, encoding a polypeptide of 560 amino acid residues with an estimated molecular mass 62.44 kDa. The SbNHX1 shows high amino acid similarity with other halophytic NHX gene and belongs to Class-I type NHXs. TMpred suggests that SbNHX1 contains 11 strong transmembrane (TM). Real time PCR analysis revealed that SbNHX1 transcript expresses maximum at 0.5 M. Transcript increases gradually by increasing the treatment duration at 0.5 M NaCl, however, maximum expression was observed at 48 h. The overexpression of SbNHX1 gene in tobacco plant showed NaCl tolerance. This study shows that SbNHX1 is a potential gene for salt tolerance, and can be used in future for developing salt tolerant crops.

The sugar beet gene encoding the sodium/proton exchanger 1 (BvNHX1) is regulated by a MYB transcription factor.

Sodium/proton exchangers (NHX) are key players in the plant response to salinity and have a central role in establishing ion homeostasis. NHXs can be localized in the tonoplast or plasma membranes, where they exchange sodium ions for protons, resulting in sodium ions being removed from the cytosol into the vacuole or extracellular space. The expression of most plant NHX genes is modulated by exposure of the organisms to salt stress or water stress. We explored the regulation of the vacuolar NHX1 gene from the salt-tolerant sugar beet plant (BvNHX1) using Arabidopsis plants transformed with an array of constructs of BvHNX1::GUS, and the expression patterns were characterized using histological and quantitative assays. The 5′ UTR of BvNHX1, including its intron, does not modulate the activity of the promoter. Serial deletions show that a 337 bp promoter fragment sufficed for driving activity that indistinguishable from that of the full-length (2,464 bp) promoter. Mutating four putative cis-acting elements within the 337 bp promoter fragment revealed that MYB transcription factor(s) are involved in the activation of the expression of BvNHX1 upon exposure to salt and water stresses. Gel mobility shift assay confirmed that the WT but not the mutated MYB binding site is bound by nuclear protein extracted from salt-stressed Beta vulgaris leaves.

Molecular and functional comparisons of the vacuolar Na+/H+ exchangers originated from glycophytic and halophytic species

A novel vacuolar Na+/H+ exchanger, CgNHX1, was cloned from a halophytic species Chenopodium glaucum by using reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) technique. Sequence alignment and phylogenetic analysis of 22 NHX genes from GenBank as well as the new CgNHX1 gene indicate that NHX genes shared a great degree of similarity, regardless of their glycophytic or halophytic origin. Expression of the CgNHX1 gene was induced by NaCl and peaked at 400 mmol/L NaCl. Overexpression of NHX1 genes in rice enhanced their tolerance to salt stress. However, there is no significant difference in salt tolerance among the transgenic rice plants overexpressing the NHX1 genes from either glycophytic or halophytic species. The Na+ content of both the wild type (WT) and transgenic plants increased when exposed to 50 and 100 mmol/L NaCl, and the Na+ concentration in transgenic plants was marginally higher than that of WT. Our data demonstrate that the overexpression of the NHX1 gene from either glycophytic or halophytic species resulted in the enhanced tolerance to salt stress at a similar level, suggesting that NHX gene per se might not be the reason accounting for the difference in salt tolerance between glycophytes and halophytes.

Comparative expression analysis of three genes from the Arabidopsis vacuolar Na+/H+ antiporter (AtNHX) family in relation to abiotic stresses

Na+/H+ antiporters (NHX) are ubiquitous transmembrane proteins that play a key role in salt tolerance of plants. In this study, the sequence of 3 Arabidopsis NHX genes (AtNHX2–4) were compared with other AtNHX members. Putative cis-elements analysis identified elements that have been associated with stress responses. The activities of the promoters AtNHX2–4 were studied in transgenic plants carrying corresponding promoter-β-glucuronidase (GUS) fusions. The AtNHX2 promoter-GUS analysis indicated that AtNHX2 was expressed in constitutive pattern with high GUS activity in roots and leaves. AtNHX2 promoter activity was not up-regulated by NaCl or abscisic acid (ABA), in contrast the AtNHX1 promoter which was previously studied. The AtNHX3 and AtNHX4 promoters showed tissue-specific activities. Strong GUS activity was detected in roots and vascular bundles of the stele in plants carrying an AtNHX4 promoter-GUS fusion, and GUS activity increased under salt stress suggesting a function related to salt tolerance. Transgenic plants carrying the AtNHX3 promoter-GUS fusion showed strong GUS activity in petals, stamens and tops of siliques, suggesting a possible role of AtNHX3 in flower and seed development. Results of histochemical analysis suggested that AtNHX2–4 are involved in divergent functions and are differentially regulated under abiotic stress. The structure of AtNHX4 was predicted to include 12 transmembrane regions and a NHX domain. Overexpression of AtNHX4 in Arabidopsis transgenic lines confers greater salt tolerance than in wild type plants. These results suggest that AtNHX4 may encode a putative vacuolar NHX that plays an important role in salt tolerance.

Arabidopsis–rice–wheat gene orthologues for Na+ transport and transcript analysis in wheat–L. elongatum aneuploids under salt stress

Lophopyrum elongatum is a wild relative of wheat that provides a source of novel genes for improvement of the salt tolerance of bread wheat. Improved Na(+) 'exclusion' is associated with salt tolerance in a wheat-L. elongatum amphiploid, in which a large proportion (ca. 50%) of the improved regulation of leaf Na(+) concentrations is controlled by chromosome 3E. In this study, genes that might control Na(+) accumulation, such as for transporters responsible for Na(+) entry (HKT1) and exit (SOS1) from cells, or compartmentalisation within vacuoles (NHX1, NHX5, AVP1, AVP2) in the model plant, Arabidopsis thaliana, were targeted for comparative analyses in wheat. Putative rice orthologues were identified and characterised as a means to bridge the large evolutionary distance between genomes from the model dicot and the more complex grass species. Wheat orthologues were identified through BLAST searching to identify either FL-cDNAs or ESTs and were subsequently used to design primers to amplify genomic DNA. The probable orthologous status of the wheat genes was confirmed through demonstration of similar intron-exon structure with their counterparts in Arabidopsis and rice. The majority of exons for Arabidopsis, rice and wheat orthologues of NHX1, NHX5 and SOS1 were conserved except for those at the amino and carboxy terminal ends. However, additional exons were identified in the predicted NHX1 and SOS1 genes of rice and wheat, as compared with Arabidopsis, indicating gene rearrangement events during evolution from a common ancestor. Nullisomic-tetrasomic, deletion and addition lines in wheat were used to assign gene sequences to chromosome regions in wheat and L. elongatum. Most sequences were assigned to homoeologous chromosomes, however, in some instances, such as for SOS1, genes were mapped to other unpredicted locations. Differential transcript abundance under salt stress indicated a complex pattern of expression for wheat orthologues that may regulate Na(+) accumulation in wheat lines containing chromosomes from L. elongatum. The identification of wheat orthologues to well characterized Arabidopsis genes, map locations and gene expression profiles increases our knowledge on the complex mechanisms regulating Na(+) transport in wheat and wheat-L. elongatum lines under salt stress.