Vacuolar NHX Research Articles

Strategies of NHX antiporters to deal with salt stress

The adverse effects of salinity on plant growth are generally associated with the low osmotic potential of the soil solution and the high level of sodium toxicity (and chlorine toxicity for some species) which cause multiple perturbations on plant metabolism, growth, and development at the molecular, biochemical and physiological levels. The vacuolar NHX and plasma membrane SOS antiporters mediate cation and proton exchange across the tonoplast and plasma membrane, respectively. The SOS transporters allow the excretion of Na+ from the cytoplasm to the outside environment and alternatively, NHXs provide Na+ transport from the cytoplasm to the vacuole. Cellular ion homeostasis is an essential phenomenon for all organisms. Most cells manage to maintain a high level of potassium and a low level of sodium in the cytoplasm through the coordination and regulation of different transporters and channels instead of the NHX-type vacuolar antiport. In this article, some important mechanisms in the regulation of ionic ions such as Na+. will be discussed.

Structure-Guided Identification of Critical Residues in the Vacuolar Cation/Proton Antiporter NHX1 from Arabidopsis thaliana.

Cation/Proton Antiporters (CPA) acting in all biological membranes regulate the volume and pH of cells and of intracellular organelles. A key issue with these proteins is their structure-function relationships since they present intrinsic regulatory features that rely on structural determinants, including pH sensitivity and the stoichiometry of ion exchange. Crystal structures are only available for prokaryotic CPA, whereas the eukaryotic ones have been modeled using the former as templates. Here, we present an updated and improved structural model of the tonoplast-localized K+, Na+/H+ antiporter NHX1 of Arabidopsis as a representative of the vacuolar NHX family that is essential for the accumulation of K+ into plant vacuoles. Conserved residues that were judged as functionally important were mutated, and the resulting protein variants were tested for activity in the yeast Saccharomyces cerevisiae. The results indicate that residue N184 in the ND-motif characteristic of CPA1 could be replaced by the DD-motif of CPA2 family members with minimal consequences for their activity. Attempts to alter the electroneutrality of AtNHX1 by different combinations of amino acid replacements at N184, R353 and R390 residues resulted in inactive or partly active proteins with a differential ability to control the vacuolar pH of the yeast.

New insights into the influence of NHX-type Cation/H+ antiporter on flower color in Phalaenopsis orchids

Vacuolar sodium/proton Na+(K+)/H+ exchanger (NHX) influence color formation because of their effects on cellular pH and Na+/K+ homeostasis. Research regarding NHXs has mainly focused on the vacuolar NHX family members. However, the NHX functions related to Phalaenopsis flower coloration remain relatively uncharacterized. In this study, we cloned and characterized PeNHX1, a vacuolar cation/H+ antiporter-encoding gene that is highly expressed in the Phalaenopsis equestris (orchid) flower lip. Phylogenetic and sequence analyses showed that PeNHX1 is a vacuolar NHX protein family member that is similar to other known vacuolar antiporters. The PeNHX1-GFP fusion protein was clearly localized to the vacuolar membrane in a transient transfection assay. A quantitative real-time PCR analysis revealed the increased expression of PeNHX1 in different flower developmental stages. Moreover, it was more highly expressed in the lip than in the other flower organs. On the basis of virus-induced gene silencing, we determined that decreased PeNHX1 expression significantly reduces P. equestris petal coloration. Furthermore, the overexpression of PeNHX1 in Phalaenopsis Big Chili caused the pH to increase and the petal color to change from red to blue. The results indicate that NHX1 may mediates the Na + or K+/H+ exchange, thereby regulating the vacuolar pH to promote blue coloration. This research provides a theoretical basis for the development of orchid varieties with blue flowers.

GhNHX3D, a Vacuolar-Localized Na+/H+ Antiporter, Positively Regulates Salt Response in Upland Cotton

Vacuolar sodium/proton (Na+/H+) antiporters (NHXs) can stabilize ion contents to improve the salt tolerance of plants. Here, GhNHX3D was cloned and characterized from upland cotton (Gossypium hirsutum). Phylogenetic and sequence analyses showed that GhNHX3D belongs to the vacuolar-type NHXs. The GhNHX3D-enhanced green fluorescent protein (eGFP) fusion protein localized on the vacuolar membrane when transiently expressed in Arabidopsis protoplasts. The quantitative real-time PCR (qRT-PCR) analysis showed that GhNHX3D was induced rapidly in response to salt stress in cotton leaves, and its transcript levels increased with the aggravation of salt stress. The introduction of GhNHX3D into the salt-sensitive yeast mutant ATX3 improved its salt tolerance. Furthermore, silencing of GhNHX3D in cotton plants by virus-induced gene silencing (VIGS) increased the Na+ levels in the leaves, stems, and roots and decreased the K+ content in the roots, leading to greater salt sensitivity. Our results indicate that GhNHX3D is a member of the vacuolar NHX family and can confer salt tolerance by adjusting the steady-state balance of cellular Na+ and K+ ions.

Beyond the patch-clamp resolution: functional activity of nonelectrogenic vacuolar NHX proton/potassium antiporters and inhibition by phosphoinositides.

We combined the patch-clamp technique with ratiometric fluorescence imaging using the proton-responsive dye BCECF as a luminal probe. Upon application of a steep cytosol-directed potassium ion (K+ ) gradient in Arabidopsis mesophyll vacuoles, a strong and reversible acidification of the vacuolar lumen was detected, whereas no associated electrical currents were observed, in agreement with electroneutral cation/H+ exchange. Our data show that this acidification was generated by NHX antiport activity, because: it did not distinguish between K+ and sodium (Na+ ) ions; it was sensitive to the NHX inhibitor benzamil; and it was completely absent in vacuoles from nhx1 nhx2 double knockout plants. Our data further show that NHX activity could be reversed, was voltage-independent and specifically impaired by the low-abundance signaling lipid PI(3,5)P2 , which may regulate salt accumulation in plants by acting as a common messenger to coordinately shut down secondary active carriers responsible for cation and anion uptake inside the vacuole. Finally, we developed a theory based on thermodynamics, which supports the data obtained by our novel experimental approach. This work, therefore, represents a proof-of-principle that can be applied to the study of proton-dependent exchangers from plants and animals, which are barely detectable using conventional techniques.

Auxin Homeostasis and Distribution of the Auxin Efflux Carrier PIN2 Require Vacuolar NHX-Type Cation/H+ Antiporter Activity.

The Arabidopsis vacuolar Na+/H+ transporters (NHXs) are important regulators of intracellular pH, Na+ and K+ homeostasis and necessary for normal plant growth, development, and stress acclimation. Arabidopsis contains four vacuolar NHX isoforms known as AtNHX1 to AtNHX4. The quadruple knockout nhx1nhx2nhx3nhx4, lacking any vacuolar NHX-type antiporter activity, displayed auxin-related phenotypes including loss of apical dominance, reduced root growth, impaired gravitropism and less sensitivity to exogenous IAA and NAA, but not to 2,4-D. In nhx1nhx2nhx3nhx4, the abundance of the auxin efflux carrier PIN2, but not PIN1, was drastically reduced at the plasma membrane and was concomitant with an increase in PIN2 labeled intracellular vesicles. Intracellular trafficking to the vacuole was also delayed in the mutant. Measurements of free IAA content and imaging of the auxin sensor DII-Venus, suggest that auxin accumulates in root tips of nhx1nhx2nhx3nhx4. Collectively, our results indicate that vacuolar NHX dependent cation/H+ antiport activity is needed for proper auxin homeostasis, likely by affecting intracellular trafficking and distribution of the PIN2 efflux carrier.

Cation Specificity of Vacuolar NHX-Type Cation/H+ Antiporters.

Cation/H+ (NHX-type) antiporters are important regulators of intracellular ion homeostasis and are critical for cell expansion and plant stress acclimation. In Arabidopsis (Arabidopsis thaliana), four distinct NHX isoforms, named AtNHX1 to AtNHX4, locate to the tonoplast. To determine the concerted roles of all tonoplast NHXs on vacuolar ion and pH homeostasis, we examined multiple knockout mutants lacking all but one of the four vacuolar isoforms and quadruple knockout plants lacking any vacuolar NHX activity. The nhx triple and quadruple knockouts displayed reduced growth phenotypes. Exposure to sodium chloride improved growth while potassium chloride was deleterious to some knockouts. Kinetic analysis of K+ and Na+ transport indicated that AtNHX1 and AtNHX2 are the main contributors to both vacuolar pH and K+ and Na+ uptake, while AtNHX3 and AtNHX4 differ in Na+/K+ selectivity. The lack of any vacuolar NHX activity resulted in no K+ uptake, highly acidic vacuoles, and reduced but not abolished vacuolar Na+ uptake. Additional K+/H+ and Na+/H+ exchange activity assays in the quadruple knockout indicated Na+ uptake that was not H+ coupled, suggesting the existence of an alternative, cation/H+-independent, Na+ conductive pathway in vacuoles. These results highlight the importance of NHX-type cation/H+ antiporters in the maintenance of cellular cation homeostasis and in growth and development.

Halophytic NHXs confer salt tolerance by altering cytosolic and vacuolar K+ and Na+ in Arabidopsis root cell

While the role of the vacuolar NHX Na+/H+ exchangers in plant salt tolerance has been demonstrated on numerous occasions, their control over cytosolic ionic relations has never been functionally analysed in the context of subcellular Na+ and K+ homeostasis. In this work, PutNHX1 and SeNHX1 were cloned from halophytes Puccinellia tenuiflora and Salicornia europaea and transiently expressed in Arabidopsis wild type Col-0 and the nhx1 mutant. Phylogentic analysis, topological prediction, analysis of evolutionary conservation, the topology structure and analysis of hydrophobic or polar regions of PutNHX1 and SeNHX1 indicated that they are unique tonoplast Na+/H+ antiporters with characteristics for salt tolerance. As a part of the functional assessment, cytosolic and vacuolar Na+ and K+ in different root tissues and ion fluxes from root mature zone of Col-0, nhx1 and their transgenic lines were measured. Transgenic lines sequestered large quantity of Na+ into root cell vacuoles and also promoted high cytosolic and vacuolar K+ accumulation. Expression of PutNHX1 and SeNHX1 led to significant transient root Na+ uptake in the four transgenic lines upon recovery from salt treatment. In contrast, the nhx1 mutant maintained a prolonged Na+ efflux and the nhx1:PutNHX1 and nhx1:SeNHX1 lines started to actively pump Na+ out of the cell. Overall, our findings suggest that PutNHX1 and SeNHX1 improve Na+ sequestration in the vacuole and K+ retention in the cytosol and vacuole of root cells of Arabidopsis, and that they interact with other regulatory mechanisms to provide a highly orchestrated regulation of ionic relations among intracellular cell compartments.

A vacuolar antiporter is differentially regulated in leaves and roots of the halophytic wild rice Porteresia coarctata (Roxb.) Tateoka.

Vacuolar NHX-type antiporters play a role in Na(+)/K(+) uptake that contributes to growth, nutrition and development. Under salt/osmotic stress they mediate the vacuolar compartmentalization of K(+)/Na(+), thereby preventing toxic Na(+)K(+) ratios in the cytosol. Porteresia coarctata (Roxb.) Tateoka, a mangrove associate, is a distant wild relative of cultivated rice and is saline as well as submergence tolerant. A vacuolar NHX homolog isolated from a P. coarctata cDNA library (PcNHX1) shows 96 % identity (nucleotide level) to OsNHX1. Diurnal PcNHX1 expression in leaves was found to be largely unaltered, though damped by salinity. PcNHX1 promoter directed GUS expression is phloem-specific in leaves, stem and roots of transgenic plants in the absence of stress. Under NaCl stress, GUS expression was also seen in the epidermal and sub-epidermal layers (mesophyll, guard cells and trichomes) of leaves, root tip. The salinity in the rhizosphere of P. coarctata varies considerably due to diurnal/semi-diurnal tidal inundation. The diurnal expression of PcNHX1 in leaves and salinity induced expression in roots may have evolved in response to dynamic changes in salinity of in the P. coarctata rhizosphere. Despite high sequence conservation between OsNHX1 and PcNHX1, the distinctive expression pattern of PcNHX1 exemplifies how variation in expression is fine tuned to suit the halophytic growth habitat of a plant.

Cloning and functional characterization of a vacuolar Na+/H+ antiporter gene from mungbean (VrNHX1) and its ectopic expression enhanced salt tolerance in Arabidopsis thaliana.

Plant vacuolar NHX exchangers play a significant role in adaption to salt stress by compartmentalizing excess cytosolic Na+ into vacuoles and maintaining cellular homeostasis and ionic equilibrium. We cloned an orthologue of the vacuolar Na+/H+ antiporter gene, VrNHX1 from mungbean (Vigna radiata), an important Asiatic grain legume. The VrNHX1 (Genbank Accession number JN656211.1) contains 2095 nucleotides with an open reading frame of 1629 nucleotides encoding a predicted protein of 542 amino acids with a deduced molecular mass of 59.6 kDa. The consensus amiloride binding motif (84LFFIYLLPPI93) was observed in the third putative transmembrane domain of VrNHX1. Bioinformatic and phylogenetic analysis clearly suggested that VrNHX1 had high similarity to those of orthologs belonging to Class-I clade of plant NHX exchangers in leguminous crops. VrNHX1 could be strongly induced by salt stress in mungbean as the expression in roots significantly increased in presence of 200 mM NaCl with concomitant accumulation of total [Na+]. Induction of VrNHX1 was also observed under cold and dehydration stress, indicating a possible cross talk between various abiotic stresses. Heterologous expression in salt sensitive yeast mutant AXT3 complemented for the loss of yeast vacuolar NHX1 under NaCl, KCl and LiCl stress indicating that VrNHX1 was the orthologue of ScNHX1. Further, AXT3 cells expressing VrNHX1 survived under low pH environment and displayed vacuolar alkalinization analyzed using pH sensitive fluorescent dye BCECF-AM. The constitutive and stress inducible expression of VrNHX1 resulted in enhanced salt tolerance in transgenic Arabidopsis thaliana lines. Our work suggested that VrNHX1 was a salt tolerance determinant in mungbean.

Potato transformation with the HvNHX3 gene and the improvement of transformant salt tolerance

Transformation by genes of various ion transporters is a tool to study plant salt tolerance mechanisms. In this work, the gene of a vacuolar NHX antiporter HvNHX3 was introduced in two potato Solanum tuberosum L. cultivars, Yubiley Zhukova and Skoroplodny-7. The binary vector pCambia-HvNHX3 was made and used for transformation. This vector carries HvNHX3 and NPTII for kanamycin selection each driven by 35S promoter from cauliflower mosaic virus. The presence of the target gene HvNHX3 and its mRNA were confirmed in 35 out of 48 kanamycin-resistant transformants. The growth parameters of 13 transgenic lines were measured in control and NaCl-containing media. The transformation of a relatively salt-tolerant cv. Yubiley Zhukova did not significantly change its growth characteristics and salt tolerance. In control conditions, most transgenic lines of cv. Skoroplodny-7 had the larger biomass and height. Unlike untransformed plants, they rooted and grew on 100 mM NaCl, indicating their greater salt tolerance.

Vacuolar Na⁺/H⁺ NHX-Type Antiporters Are Required for Cellular K⁺ Homeostasis, Microtubule Organization and Directional Root Growth.

Na+/H+ antiporters (NHXs) are integral membrane transporters that catalyze the electroneutral exchange of K+ or Na+ for H+ and are implicated in cell expansion, development, pH and ion homeostasis and salt tolerance. Arabidopsis contains four vacuolar NHX isoforms (NHX1–NHX4), but only the functional roles for NHX1 and NHX2 have been assessed thus far. Colocalization studies indicated that NHX3 and NHX4 colocalize to the tonoplast. To investigate the role of all vacuolar NHX isoforms, a quadruple knockout nhx1nhx2nhx3nhx4, lacking all vacuolar NHXs, was generated. Seedlings of nhx1nhx2nhx3nhx4 displayed significantly reduced growth, with markedly shorter hypocotyls. Under high K+, but not Na+, pronounced root skewing occurred in nhx1nhx2nhx3nhx4, suggesting that the organization of the cytoskeleton might be perturbed. Whole mount immunolabeling of cortical microtubules indicated that high K+ caused significant microtubule reorganization in nhx1nhx2nhx3nhx4 root cells of the elongation zone. Using microtubule stabilizing (Taxol) and destabilizing (propyzamide) drugs, we found that the effect of K+ on nhx1nhx2nhx3nhx4 root growth was antagonistic to that of Taxol, whereas elevated K+ exacerbated the endogenous effect of propyzamide on root skewing. Collectively, our results suggest that altered K+ homeostasis leads to an increase in the dynamics of cortical microtubule reorganization in nhx1nhx2nhx3nhx4 root epidermal cells of the elongation zone.

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.

Cellular ion homeostasis: emerging roles of intracellular NHX Na+/H+ antiporters in plant growth and development

Recent evidence highlights novel roles for intracellular Na(+)/H(+) antiporters (NHXs) in plants. The availability of knockouts and overexpressors of specific NHX isoforms has provided compelling genetic evidence to support earlier physiological and biochemical data which suggested the involvement of NHX antiporters in ion and pH regulation. Most plants sequenced to date contain multiple NHX members and, based on their sequence identity and localization, can be grouped into three distinct functional classes: plasma membrane, vacuolar, and endosomal associated. Orthologues of each functional class are represented in all sequenced plant genomes, suggesting conserved and fundamental roles across taxa. In this review we seek to highlight recent findings which demonstrate that intracellular NHX antiporters (i.e. vacuolar and endosomal isoforms) play roles in growth and development, including cell expansion, cell volume regulation, ion homeostasis, osmotic adjustment, pH regulation, vesicular trafficking, protein processing, cellular stress responses, as well as flowering. A significant new discovery demonstrated that in addition to the better known vacuolar NHX isoforms, plants also contain endosomal NHX isoforms that regulate protein processing and trafficking of cellular cargo. We draw parallels from close orthologues in yeast and mammals and discuss distinctive NHX functions in plants.

Homology Modeling and Structural Analysis of NHX Antiporter of Leptochloa fusca (L.)

Eukaryotic NHX transporters are trans-membrane proteins which are vital for cellular homeostasis. Plants vacuolar NHX antiporters have been suggested to be involved in sequestering Na+ into vacuoles, thus preventing toxic effects of Na+ in the cytoplasm. Primary and secondary structure analysis of NHX antiporter of Leptochloa fusca suggested that this antiporter is a hydrophobic protein containing a significant proportion of alpha helixes. In this study, a three-dimensional structures of NHX antiporter predicted by in silico 3D homology modeling studies. The resulting model was refined by energy minimization, subjected to the quality assessment from both geometric and energetic aspects and was found to be of reasonable quality. Validation of 3D structure was done by plotting Ramachandran plot and calculation of QMEAN score. This predicted information will help in better understanding of mechanisms underlying to salt tolerance in monocot plants and use of this information in protein engineering to improve plants to high salinity conditions.

Conserved and Diversified Gene Families of Monovalent Cation/H+ Antiporters from Algae to Flowering Plants

All organisms have evolved strategies to regulate ion and pH homeostasis in response to developmental and environmental cues. One strategy is mediated by monovalent cation–proton antiporters (CPA) that are classified in two superfamilies. Many CPA1 genes from bacteria, fungi, metazoa, and plants have been functionally characterized; though roles of plant CPA2 genes encoding K+-efflux antiporter (KEA) and cation/H+ exchanger (CHX) families are largely unknown. Phylogenetic analysis showed that three clades of the CPA1 Na+–H+ exchanger (NHX) family have been conserved from single-celled algae to Arabidopsis. These are (i) plasma membrane-bound SOS1/AtNHX7 that share ancestry with prokaryote NhaP, (ii) endosomal AtNHX5/6 that is part of the eukaryote Intracellular-NHE clade, and (iii) a vacuolar NHX clade (AtNHX1–4) specific to plants. Early diversification of KEA genes possibly from an ancestral cyanobacterium gene is suggested by three types seen in all plants. Intriguingly, CHX genes diversified from three to four members in one subclade of early land plants to 28 genes in eight subclades of Arabidopsis. Homologs from Spirogyra or Physcomitrella share high similarity with AtCHX20, suggesting that guard cell-specific AtCHX20 and its closest relatives are founders of the family, and pollen-expressed CHX genes appeared later in monocots and early eudicots. AtCHX proteins mediate K+ transport and pH homeostasis, and have been localized to intracellular and plasma membrane. Thus KEA genes are conserved from green algae to angiosperms, and their presence in red algae and secondary endosymbionts suggest a role in plastids. In contrast, AtNHX1–4 subtype evolved in plant cells to handle ion homeostasis of vacuoles. The great diversity of CHX genes in land plants compared to metazoa, fungi, or algae would imply a significant role of ion and pH homeostasis at dynamic endomembranes in the vegetative and reproductive success of flowering plants.

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.

Rôles biologiques des antiports vacuolaires NHX : acquis et perspectives d’amélioration génétique des plantes

NHX antiporters are membrane proteins that mediate cation and proton exchanges across vacuolar membranes. In particular, they catalyze the Na+–H+exchanges and, consequently, sequester Na+into vacuoles. The latter mechanism protects essential enzymatic reactions in the cytoplasm from toxicity due to excess Na+levels, while adjusting turgor by means of vacuolar Na+as an osmoticum. The function of vacuolar NHX antiporters in plants has been studied primarily in the context of salinity tolerance. The expression of induction of NHX-type genes in response to NaCl treatment was observed for most NHX-type genes characterized, and their overexpression allowed to confer an improved tolerance to salinity, initially in transgenic model plants ( Arabidopsis thaliana (L.) Heynh. and tobacco) and, subsequently, in cultivated plants (tomato, rice, wheat, etc.). As many genes encoding these antiporters have been cloned from both salt-sensitive and salt-tolerant species and because multiple isoforms can be identified in genomes that have been sequenced, it is likely that these antiporters assume functions at various levels beside providing tolerance to salinity. Indeed, in addition to playing a role in other known functions such as pH regulation, ion homeostasis, and osmotic and turgor regulation, they also have an important role in diverse physiological processes, including control of cell growth and proliferation, vesicle biogenesis and trafficking, protein targeting, and more specifically, leaf development, blue-flower coloration, and grape berry maturation. If we take into account the high probability that vacuolar NHX antiporters play a role in multiple physiological processes, it would be possible, by exploiting the available information, to use several molecular methods, such as overexpression or silencing of NHX-type genes, to achieve the selection and improvement of interesting plant characters, including flower coloration, fruit maturation, and tolerance to salinity and drought.

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.

A Grape Berry (Vitis vinifera L.) Cation/Proton Antiporter is Associated with Berry Ripening

We have cloned and characterized VvNHX1, a gene encoding a vacuolar cation/H(+) antiporter from Vitis vinifera cv. Cabernet Sauvignon. VvNHX1 belongs to the vacuolar NHX protein family and showed high similarity to other known vacuolar antiporters. The expression of VvNHX1 partially complements the salt- and hygromycin-sensitive phenotypes of an ena1-4 nhx1 yeast strain. Immunoblots of vacuoles of yeast expressing a VvNHX1, together with the expression of a VvNHX1-GFP (green fluorescent protein) chimera demonstrated that VvNHX1 localized to the vacuoles. VvNHX1 displayed low affinity K(+)/H(+) and Na(+)/H(+) exchange activities (12.8 and 40.2 mM, respectively). The high levels of expression of VvNHX1 during the véraison and post-véraison stages would indicate that the increase in vacuolar K(+) accumulation, mediated by VvNHX1, is needed for vacuolar expansion. This process, together with the rapid accumulation of reducing sugars, would drive water uptake to the berry and the concomitant berry size increase, typical of the post-véraison stage of growth.