Cloning and Characterization of the Na^+/H^+ Antiport Genes from Triticum aestivum

The Na+/H+ antiport genes namedTaNHX1andTaNHX2were cloned by screening a salt_stressed wheat cDNA library using rice Na+/H+ antiport cDNA fragment as the probe. Sequencing analysis showed thatTaNHX1was 2 029 bp in length and contained a complete ORF of 1 638 bp. TheTaNHX1encodes a polypeptide of 546 amino acids with a transmembrane domain DIFFIYLLPPI.TaNHX2was 1 693 bp in length consisting of a partial ORF followed by a 3′_UTR of 808 bp. The amino acid sequence of these two genes were about 70% identical to the known NHX genes from rice, Arabidopsis and Atriplex. A RT_PCR assay showed that the level ofTaNHX1transcripts was increased and reached a steady higher level in the seedlings after 3 h treatment with 400 mmol/L NaCl.

Screening an Na^+/H^+ Antiporter Gene from the Halophiles Colonizing in the Dagong Ancient Brine Well of Zigong City,China

[Objective] This study aimed to screen an Na＋/H＋ antiporter gene from the halophiles colonizing in the Dagong Ancient Brine Well in Zigong City, China, and then analyze the gene structure and properties of the protein encoded by this gene. [Method] Metagenomic DNA libraries of halophiles from the Dagong Ancient Brine Well were used for screening genes with Na＋/H＋ antiporter activity in antiporter-defi- cient E. coil KNabc strain by functional complementation. Then the start codon, stop codon, ORF, -35 region, -10 region and SD sequence of Na~/H＋ antiporter gene, as well as the molecular weight, isoelectric point, hydrophobic region, transmembrane domain, phyletic evolution and salt resistance of protein encoded by the gene were investigated. [Result] A new Na＋/H＋ antiporter gene m-nha was obtained, which ,ren- dered the antiporter-negative mutant E. coil KNabc cells with both the resistance to Na＋ and the ability to grow under alkaline conditions. [Conclusion] The structure and amino acid sequence of M-Nha was different from the previously reported Na＋/H~ antiporters, and the m-nha gene disclosed from the Dagong Ancient Brine Well was identified as a novel Na＋/H＋ antiporter gene. This study was significant not only in helping us understand the salt tolerance of halophiles in ancient brine wells and develop and utilize the genes resource, but also in exploring new salt-tolerant genes.

渗透胁迫调节基因——Na^+/H^+ Antiporter基因与植物耐盐性

Na+/H+Antiporter基因与植物耐盐性密切相关,其编码产物Na+/H+逆向转运蛋白通过Na+外排和Na+区隔化来维持植物细胞内较低的Na+水平,降低Na+的毒害,从而对植物的耐盐性起重要作用。

Cloning and Identification of A New Na^+/H^+ Antiporter Gene ZmSOS1 in Maize(Zea mays L.)

[ Objective] The study aimed to clone and identify Na^＋/H^＋ antiporter genes in maize, and provided the information for characterizing the function of such genes in abiotic stress tolerance of maize. Method The in silico cloning, RT-PCR, and bioinformatics analysis were used in this study. Result By in sifico cloning, a plasma membrane Na^＋/H^＋ antiporter gene, named as ZmSOS1 （EMBL accession No. BN001309）, was cloned from maize （ Zea mays L. ）. ZmSOS1 has an open reading frame （ORF） of 3 411 bp which encoded a protein of 1 136 amino acids. By multiple sequence alignment analysis, it showed the predicated peptide of ZmSOS1 were 61% and 82% identities in amino acids to the plasma membrane Na^＋/H^＋ antiporter AtSOS1 and OsSOS1, respectively. The RT-PCR analysis revealed that ZmSOS1 could be significantly up-regulated by salt stress, which indicated ZmSOS1 might play a role in salt tolerance of maize. Conclusion ZmSOS1 is a putative plasma membrane Na^＋/H^＋ antiporter gene and may play a role in abiotic stress tolerance of maize.

Involvement of Plasma Membrane Ca^(2+)/H^+ Antiporter in Cd^(2+) Tolerance

Cation exchangers （CAXs） belong to the cation/Ca2＋exchanger superfamily which have been extensively investigated in plant tonoplasts over the last decade. Recently, the roles of CAXs involved in heavy metal accumulation and tolerance in plants have been studied for phytoremediation and food security. In this mini review, we summarize the roles of the Ca2＋/H＋ antiporter in Ca2＋ signal transduction, maintaining ion homeostasis and sequestering heavy metals into the vacuole. Moreover, we present a possible role of the plasma membrane Ca2＋/H＋ antiporter in heavy metal detoxification.

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 Gm KEAs genes and 10 soybean Gm NHXs 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 Na Cl 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.

Effects of NaCl and Iso-Osmotic Polyethylene Glycol Stress on Na^+/H^+Antiport Activity of Three Malus species with Different Salt Tolerance

Salt stress contains osmotic and ionic stress, while iso-osmotic polyethylene glycol （PEG） has only osmotic stress. This study aimed to compare the different effects on the activity of H＋-ATPase, proton pump and Na＋/H＋antiport in Malus seedlings between osmotic and ionic stress. Species of salt tolerant Malus zumi, middle salt tolerant Malus xiaojinensis and salt sensitive Malus baccata were used as experimental materials. Malus seedlings were treated with NaCl and iso-osmotic PEG stress. The activity of H＋-ATPase, proton pump and Na＋/H＋antiport of plasmolemma and tonoplast in Malus seedlings were obviously increased under salt stress, and those in salt-tolerant species increased more. Under the same NaCl concentration, the activity of H＋-ATPase, proton pump and Na＋/H＋antiport of plasmolemma and tonoplast in salt-tolerant species were all obviously higher than those in salt-sensitive one. Higher Na＋/H＋antiport activity of plasmolemma and tonoplast in salt-tolerant species could help to extrude and compartmentalize sodium in roots under salt stress. The ascent rate of activity of H＋-ATPase, proton pump and Na＋/H＋antiport in Malus seedlings under the three salt concentration stress was all obviously higher than that under the iso-osmotic PEG stress. It indicated that the sodium ion effect had more stimulation on the activity of H＋-ATPase, proton pump and Na＋/H＋antiport in salt-tolerant species, and salt-tolerant species has higher capability of sodium extrusion and compartmentalization in roots and is therefore more salt tolerant.

Expression analysis of a Na^+/H^+ antiporter gene PeNHX1 from Populus euphratica

Na+/H+ antiporters play an important role in the salt tolerance of a wide variety of plants.Using the rapid amplification of cDNA ends method,a Na+/H+ antiporter gene (PeNHX1) was isolated from Populus euphratica.The deduced amino acid sequence contained 528 amino acid residues with a conserved amiloride-binding domain (77LFFIYLLPPI86) and shared more than 68% identity with that of AtNHX1 from Arabidopsis thaliana.PeNHX1 can confer resistance to Na+,as well as Li+,to (EP432) an Escherichia coli strain deficient in both nhaA and nhaB,thus proving that it is a functional Na+/H+ antiporter.PeNHX1 expression profile in EP432 reflected pH independent manner.PeNHX1 expression was regulated by salt at the transcriptional level.Meanwhile,results demonstrated that transcripts of PeNHX1 in P.euphratica calli showed a salt dependent response,and thus provide a valuable tool for studying signaling and biochemical pathways involved in salt recognition and response in P.euphratica.

Cloning of Na^+/H^+ antiport gene from Dunaliella salina

1 Introduction Dunaliella Salina,which taxi Dunaliella,Volvocales,Chlorophyceae Chlorophyta,is unicell algae with double flagllum at top,and cup shaped chloroplast without cell wall.Dunaliella Salina is the most salt tolerance eucaryotes.It can grow at the range of salt concentration

Role of Na^+/H^+ antiporter in pulmonary artery smooth muscle of hypoxic pulmonary hypertension in the rat

Objective: To investigate the role of Na+ /H+ antiporter in the hypoxic pumonary hypertension ofrats. Methods: Thirty Wistar rats were randomly divided into 3 groups with 10 in each group: controlgroup, 3--week hypoxia group and 8--week hypoxia group. After the isolation of pulmonary artery smoothmuscles, pHi was determined by fluorescence measurement of the pH--sensitive dye BCECF and theexpression of NHE--1 mRNA was detected with reverse transcription--polymerase chain reaction. Results: ThepHi and expression of NHE-1 mRNA of pulmonary artery smooth muscle cell in the hypoxia groups weresignificantly increased than those in the normal group (P < 0. 01 ). There was no remarkable differencebetween the hypoxia groups. Conclusion: With the function of regulation pHi., NHE--1 may play an importantrole in the pulmonary vascular remodeling of pulmonary hypertension. The result provides a new therapeuticmethod with NHE--1 inhibitors and/or gene therapy for the hypoxic pulmonary hypertension.

Effect of salt stress on the expression of NHX-type Na^+/H^+ antiporters in Populus euphratica and P.pruinosa calli

Populus euphratica and Populuspruinosa, sister species in the Turanga Section （Salicaceae）, growing in semi-arid saline areas are known for their high salinity tolerance. In this study, by combining growth level with Na＋ and K＋ contents, the expression level of vacuolar Na＋/H＋ antiporters was investigated for NaCl-induced changes in P. euphratica and t3. pru- inosa calli. Compared to R euphratica, P. pruinosa calli grew well in 200 mM NaC1 stress from 14. to 21 days. Increasing the stressed time caused an increase in Na＋ content concomitant with a decrease in K＋ content in P. euphratica calli, whereas, with the presence of 200 mM NaCI, K＋ content has a less increase in 14 and 21 days than in 7 days which was detected in R pruinosa calli. The transcript levels of six genes coding for NHX-type Na＋/H＋ antiporters suggest that vacuolar NHX1-NHX6 antiporters play important roles in responding to salt stress in R pruinosa. Our data suggest that there exists a higher salt tolerance for P. pruinosa than P. euphratica at the cellular level, Na＋ avoidance or accumulation is observed in cellular compartments, and that expression of NHX antiporters is linked to the accumulator phenotype.

Cloning of Three Antiporter Genes from Arabidopsis and Rice for Over-Expressing Them in Farmer Popular Tomato Varieties of Bangladesh

Salinity is one of the most critical environmental problems, which causes plant growth retardation by disturbing intracellular ion homeostasis. The Na+/H+ antiporter plays an important role in resistance to salt stress by sequestering Na+ in exchange for H+ across the vacuolar membranes. In the current study, the coding regions of two Arabidopsis antiporters (AtNHX1 and AtNHX2) and one rice antiporter (OsNHX1) were amplified by target specific PCR. PCR amplicons were first cloned into pENTR/D-TOPO and later recombined with a destination vector (pK7WG2.0) by LR reaction. Positive clones were selected by PCR, restriction digestion (RD) and sequencing. They were then transformed into Agrobacterium tumefaciens (LBA4404 strain) for subsequent transformation of farmer popular tomato varieties.

Plant and Yeast NHX Antiporters: Roles in Membrane Trafficking

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 ScNhxlp, 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.

Dynamic changes of phosphatidylinositol and phosphatidylinositol 4-phosphate levels modulate H^(+)-ATPase and Na^(+)/H^(+) antiporter activities to maintain ion homeostasis in Arabidopsis under salt stress

Plant metabolites are dynamically modified and distributed in response to environmental changes.How-ever,it is poorly understood how metabolic change functions in plant stress responses.Maintaining ion ho-meostasis under salt stress requires coordinated activation of two types of central regulators:plasma membrane(PM)H^(+)-ATPase and Na^(+)/H^(+) antiporter.In this study,we used a bioassay-guided isolation approach to identify endogenous small molecules that affect PM H^(+)-ATPase and Na^(+)/H^(+) antiporter activities and identified phosphatidylinositol(PI),which inhibits PM H^(+)-ATPase activity under non-stress conditions in Arabidopsis by directly binding to the C terminus of the PM H^(+)-ATPase AHA2.Under salt stress,the phosphatidylinositol 4-phosphate-to-phosphatidylinositol(PI4P-to-PI)ratio increased,and PI4P bound and activated the PM Na^(+)/H^(+) antiporter.PI prefers binding to the inactive form of PM H^(+)-ATPase,while PI4P tends to bind to the active form of the Na^(+)/H^(+) antiporter.Consistent with this,pis1 mutants,with reduced levels of PI,displayed increased PM H^(+)-ATPase activity and salt stress toler-ance,while the pi4kβ1 mutant,with reduced levels of PI4P,displayed reduced PM Na^(+)/H^(+) antiporter activity and salt stress tolerance.Collectively,our results reveal that the dynamic change between PI and PI4P in response to salt stress in Arabidopsis is crucial for maintaining ion homeostasis to protect plants from un-favorable environmental conditions.

Ectopic Expression of a Bacterium NhaD-type Na^+/H^+ Antiporter Leads to Increased Tolerance to Combined Salt/Alkali Stresses

AaNhaD, a gene isolated from the soda lake alkaliphile Alkalimonas amylolytica, encodes a Na＋/H＋ antiporter crucial for the bacterium＇s resistance to salt/alkali stresses. However, it remains unknown whether this type of bacterial gene may be able to increase the tolerance of flowering plants to salt/alkali stresses. To investigate the use of extremophile genetic resources in higher plants, transgenic tobacco BY-2 cells and plants harboring AaNhaDwere generated and their stress tolerance was evaluated. Ectopic expression of AaNhaD enhanced the salt tolerance of the transgenic BY-2 cells in a pH-dependent manner. Compared to wild-type controls, the transgenic cells exhibited increased Na＋ concentrations and pH levels in the vacuoles. Subcellular localization analysis indicated that AaNhaD-GFP fusion proteins were primarily localized in the tonoplasts. Similar to the transgenic BY-2 cells, AaNhaD.overexpressing tobacco plants displayed enhanced stress tolerance when grown in saline-alkali soil. These results indicate that AaNhaD functions as a pH-dependent tonoplast Na＋/H＋ antiporter in plant cells, thus presenting a new avenue for the genetic improvement of salinity/alkalinity tolerance.

Effect of fatty acids on poly-amine contents and Na^+/H^+ antiport activity in PM vesicles isolated from roots of barley under salt stress

With 200 mmol/L NaCl treatment on barley cultivar 'Jian 4' (Hordeum vulgare L. cv. J4) seedlings for 6 d, the contents of covalently and noncovalently conjugated polyamines (PAs) and activities of H+-ATPase in plasma membrane (PM) vesicles isolated from the roots decreased remarkably. Moreover, the activity of Na+/H+ antiport was detected first in PM vesicles. The results showed that the decrease in the contents of membrane phospholipid, noncovalently conjugated PAs and activity of H+-ATPase caused by NaCl could be restored partially by application of 1 mmol/L stearic acid (C16:0) and linoleic acid (C18:2), and C18:2 was more effective than C16:0. In addition, a reduction in the contents of covalently conjugated PAs was only reversed partially in the presence of C18:2. Furthermore, Na+/H+ antiport activity was strengthened by exogenous C16:0 and C18:2, and C18:2 was more effective than C16:0. The correlative analysis suggested that, after application of C16:0 and C18:2 under salt stress, there was a

A Putative Chloroplast-Localized Ca2＋/H＋ Antiporter CCHA1 Is Involved in Calcium and pH Homeostasis and Required for PSII Function in Arabidopsis

Calcium is important for chloroplast, not only in its photosynthetic but also nonphotosynthetic functions. Mul- tiple Ca2＋/H＋ transporters and channels have been described and studied in the plasma membrane and organ- elle membranes of plant cells; however, the molecular identity and physiological roles of chloroplast Ca2＋/H＋ antiporters have remained unknown. Here we report the identification and characterization of a member of the UPFO016 family, CCHA1 （a chloroplast-localized potential Ca2＋/H＋ antiporter）, in Arabidopsis thaliana. We observed that the ccha I mutant plants developed pale green leaves and showed severely stunted growth along with impaired photosystem II （PSII） function. CCHA1 localizes to the chloroplasts, and the levels of the PSII core subunits and the oxygen-evolving complex were significantly decreased in the ccha I mutants compared with the wild type. In high Ca2＋ concentrations, Arabidopsis CCHA1 partially rescued the growth defect of yeast gdtl3 null mutant, which is defective in a Ca2＋/H＋ antiporter. The cchal mutant plants also showed significant sensitivity to high concentrations of CaCI2 and MnCI2, as well as variation in pH. Taken these results together, we propose that CCHA 1 might encode a putative chloroplast-localized Ca2＋/H＋ antiporter with critical functions in the regulation of PSII and in chloroplast Ca2＋ and pH homeostasis in Arabidopsis.

Plastid KEA-type cation/H+ antiporters are required for vacuolar protein trafficking in Arabidopsis

Arabidopsis plastid antiporters KEA1 and KEA2are critical for plastid development, photosynthetic efficiency, and plant development.Here, we show that KEA1 and KEA2 are involved in vacuolar protein trafficking. Genetic analyses found that the kea1 kea2 mutants had short siliques, small seeds, and short seedlings. Molecular and biochemical assays showed that seed storage proteins were missorted out of the cell and the precursor proteins were accumulated in kea1 kea2. Protein storage vacuoles(PSVs) were smaller in kea1 kea2. Further analyses showed that endosomal trafficking in kea1 kea2 was compromised. Vacuolar sorting receptor 1(VSR1) subcellular localizations, VSR–cargo interactions, and p24 distribution on the endoplasmic reticulum(ER) and Golgi apparatus were affected in kea1 kea2. Moreover, plastid stromule growth was reduced and plastid association with the endomembrane compartments was disrupted in kea1 kea2. Stromule growth was regulated by the cellular pH and K+homeostasis maintained by KEA1 and KEA2. The organellar pH along the trafficking pathway was altered in kea1 kea2. Overall, KEA1 and KEA2 regulate vacuolar trafficking by controlling the function of plastid stromules via adjusting pH and K+homeostasis.

Identification of H^+/Ca^(2+) Antiporter in Barley Root Plasma Membrane

On the basis of two types of calcium transport system detected in the barley root plasma membrane,the mechanisms of the calcium transport have been further studied.Ionophore CCCP has been found to inhibit Mg^(2+) -dependent calcium transport by 20%.In contrast,Mg^(2+) -independent calcium trans- port is insensitive to CCCP.The Mg^(2+) -dependent calcium transport following the collapse of H^+ gradient across the plasma membrane could be driven by the H^+ gradient either set up by ATP or imposed artificially. Any relation between Mg^(2+) -independent calcium transport and H^+ gradient has not been observed.These results indicate that Mg^(2+) -dependent calcium transport is accompanied by the decrease of H^+ gradient,and Mg^(2+) -independent calcium transport has nothing to do with the H^+ gradient.It is therefore suggested that the calcium transport across the barley root plasma membrane is driven by ATPase that is independent of Mg^(2+),and H^+/Ca^(2+) antiporter that is dependent on Mg^(2+).

Molecular characterization of GmNHX2,a Na^+/H^+ antiporter gene homolog from soybean,and its heterologous expression to improve salt tolerance in Arabidopsis

Na+/H+ antiporters have been well documented to enhance plant salt tolerance by regulating cellular ion homeostasis. Here, a putative Na+/H+ antiporter gene homolog GmNHX2 from soybean was cloned and predicted to encode a protein of 534 amino acids with 10 putative transmembrane domains. GmNHX2 was expressed in all soybean plant tissues but enriched in roots and its expression was induced by NaCl and polyethylene glycol (PEG) treatments. GmNHX2 exhibits greater sequence similarity with LeNHX2 and AtNHX6 than that of AtNHX1 and AtSOS1. Although phylogenetic analysis clustered GmNHX2 with organellar (tonoplast and vesicles) antiporters, the GmNHX2-EGFP (enhanced green fluorescent protein) fusion protein was possibly localized in the plasma membrane or organelle membrane of transgenic plant cells. Furthermore, transgenic Arabidopsis plants expressing GmNHX2 were more tolerant to high NaCl concentrations during germination and seedling stages when compared with wild-type plants. These results suggest that GmNHX2 is a membrane Na+/H+ antiporter and may function to regulate ion homeostasis under salt stress.