Antiporter Gene from Hordum brevisubulatum （Trin.） Link and Its Overexpression in Transgenic Tobaccos

A vacuolar Na^ /H^ antiporter cDNA gene was successfully isolated fromHordeum brevisubulatum (Trin.) Link using the rapid amplification ofcDNA ends (RACE) method. The gene was named HbNHXI and was found to consist of 1 916 bp encoding a predicted polypeptide of 540 amino acids with a conserved amiloride-binding domain. Phylogenetic tree analysis of the Na^ /H^ antiporters showed that the HbNHXI gene shares 55.3%-74.8% similarity with the vacuolar-type Na^ /H^ antiporters. Transgenic tobaccos that contain the HbNHXI gene, integrated by forward insertion into the tobacco genome, were obtained via Agrobacterium tumerfaciens and characterized for the determination of the concentration of Na^ and K^ ions, as well as proline, in the presence of 300 mmol/L NaCl. The T1 transgenic plants showed more tolerance to salt and drought than did wild-type plants. Our data suggest that overexpression of the HbNHXI gene could improve the tolerance of transgenic tobaccos to salt and drought through the function of the vacuolar Na^ /H^ antiporter.     

The transgene pyramiding tobacco with betaine synthesis and heterologous expression of AtNHX1 is more tolerant to salt stress than either of the tobacco lines with betaine synthesis or AtNHX1

Previous studies have shown that the overexpression of betA (encoding choline dehydrogenase from Escherichia coli ) or AtNHX1 (a vacuolar Na⁺/H⁺ antiport from Arabidopsis thaliana ) gene can improve the salt tolerance of transgenic plants. However, little is known about the effects of the transgene pyramiding of betA and AtNHX1 . Here, betA  +  AtNHX1 transgene pyramiding tobacco was produced by sexual crossing, and the salt tolerance was evaluated at the cellular and plant levels. In NaCl stress, the Na⁺ concentration in vacuoles and vacuolar membrane potential of transgene pyramiding cells were similar to those of AtNHX1 -transgenics, and much higher than those of betA -transgenics when detected using fluorescent dye staining; transgene pyramiding cells showed a higher protoplast viability and comparable mitochondrial activity as compared with single transgenics; and transgene pyramiding plants showed comparable Na⁺ content in leaves as compared with AtNHX1 -transgenics and remarkably higher than betA -transgenics; and transgene pyramiding lines exhibited higher percentage of seed germination, better seedling growth and higher fresh weight than lines that had betA or AtNHX1 alone. Based on the integrative analysis of salt tolerance, the consistency between the cellular level and the whole plant level was confirmed and the transgene pyramiding plants exhibited improved salt tolerance, but compared with the plants with betA or AtNHX1 alone, the differences were relatively small. Other mechanisms involved in salt tolerance should be considered to further enhance transgene pyramiding plants salt tolerance.     

Sucrose and ouabain effects on the kinetic properties of a membrane-bound (Na++ K++ Mg2+) ATPase in sugar beet roots

Kinetic studies of a microsomal (Na⁺+ K⁺+ Mg²⁺)ATPase from sugar beet roots ( Beta vulgaris L. cv. Monohill) show that sucrose influences the MgATPase in different ways depending on the presence of K⁺ and/or Na⁺ 1) In the presence of the substrate MgATP and Na⁺ the effect of sucrose follows simple Michaelis-Menten kinetics. 2) In the presence of substrate together with K⁺ or (K⁺+ Na⁺), sucrose has little effect on the ATPase activity. 3) In the presence of Na⁺, onabain acts as an uncompetitive inhibitor with respect to MgATP. 4) In the presence of K⁺ or (K⁺+ Na⁺), the inhibition by ouabain is somewhat depressed and shows non-linearity when 1/v is plotted versus 1/MgATP. 5) Sucrose and Na⁺ activate in a competitive way, so that a successive increase of the Na⁺ level decreases the activation by sucrose. Both K_m and V-values are thereby changed. 6) The sucrose activation in the presence of Na⁺ is also influenced by ouabain. It is, therefore, suggested that Na⁺ may regulate the interference between the Na⁺/K⁺ pump and a sucrose sensitive system.     

The involvement of a vanadate-sensitive ATPase in plasma membranes of a salt tolerant cyanobacterium

Plasma membranes of the marine cyanobacterium Spirulina subsalsa were tested for ATPase activity, and for involvement in salt stress. Transition of cells from saline to hypersaline medium enhances the respiratory activity associated with extrusion of Na⁺ and Cl⁻, and persisting salt stress induces synthesis of respiratory enzymes in the plasma membranes. The membranes possess an ATPase, specific for ATP and Mg²⁺ and sensitive to orthovanadate and dicyclohexylcarbodiimide. Immunoblot analysis of plasma membrane polypeptides from Spirulina subsalsa with anti- Arabidopsis H⁺-ATPase serum identified a single polypeptide of 100 kDa, which cross-reacted with the antibodies. An unusual feature of this ATPase is a specific stimulation by Na⁺ ions. Prolonged adaptation of S. subsals cells to hypersaline conditions induced an increase in ATPase activity in subsequent plasma membrane preparations, as well as a higher content of the 100 kDa polypeptide. It is suggested that the ATPase investigated is an H⁺-pump, which is involved in extrusion of Na⁺ and in conferring resistance to salt stress.     

The HAL1 function on Na+ homeostasis is maintained over time in salt-treated transgenic tomato plants, but the high reduction of Na+ in leaf is not associated with salt tolerance

To achieve a deeper knowledge on the function of HAL1 gene in tomato ( Solanum lycopersicum ) plants submitted to salt stress, in this study, we studied the growth and physiological responses to high salt stress of T3 transgenic plants (an azygous line without transgene and both homozygous and hemizygous lines for HAL1 ) proceeding from a primary transformant with a very high expression level of HAL1 gene. The homozygous plants for HAL1 gene did not increase their salt tolerance in spite of an earlier and higher reduction of the Na⁺ accumulation in leaves, being moreover the Na⁺ homeostasis maintained throughout the growth cycle. The greater ability of the homozygous line to regulate the Na⁺ transport to the shoot to long term was even shown in low accumulation of Na⁺ in fruits. By comparing the homozygous and hemizygous lines, a higher salt tolerance in the hemizygous line, with respect to the homozygous line, was observed on the basis of fruit yield. The Na⁺ homeostasis and osmotic homeostasis were also different in homozygous and hemizygous lines. Indeed, the Na⁺ accumulation rate in leaves was greater in hemizygous than in homozygous line after 35 days of 100 m M NaCl treatment and only at the end of growth cycle did the hemizygous line show leaf Na⁺ levels similar to those found in the homozygous line. With respect to the osmotic homeostasis, the main difference between lines was the different contribution of inorganic and organic solutes to the leaf osmotic balance. Taken together, these results suggest that the greater Na⁺ exclusion ability of the homozygous line overexpressing HAL1 induces a greater use of organic solutes for osmotic balance, which seems to have an energy cost and hence a growth penalty that reverts negatively on fruit yield.     

Na+/H+ antiport activity in tonoplast vesicles from roots of the salt-tolerant Plantago maritima and the salt-sensitive Plantago media

Plantago species differ in their strategy towards salt stress, a major difference being the uptake and distribution of Na⁺ ions. A salt-sensitive ( Plantago media L.) and a salt-tolerant ( P. maritima L.) species were compared with respect to Na⁺/H⁺ antiport activities at the tonoplast. After exposure of the plants to 50 m M NaCl for 6 days isolated tonoplast vesicles of P. maritima showed Na⁺/H⁺ antiport activity with saturation kinetics and a K_m of 2.4 m M Na⁺, NaCl-grown P. media and the control plants of both species showed no antiport activity. Selectivity of the antiport system for Na⁺ was high and was determined by adding different chloride salts after formation of a Δ pH in the vesicles. Specific tonoplast ATPase activities were similar in the two species and did not alter after exposure to NaCl stress.     

Aquaporin functionality in relation to H+-ATPase activity in root cells of Capsicum annuum grown under salinity

As water and nutrient uptake should be related in the response of plants to salinity, the aim of this paper is to establish whether or not aquaporin functionality is related to H⁺-ATPase activity in root cells of pepper ( Capsicum annuum L.) plants. Thus, H⁺-ATPase activity was measured in plasma membrane vesicles isolated from roots and aquaporin functionality was measured using a cell pressure probe in intact roots. Salinity was applied as 60 m M NaCl or 60 m M KCl, to determine which ion (Na⁺, K⁺ or Cl⁻) is producing the effects. We also investigated whether the effects of both salts were ameliorated by Ca²⁺. Similar results were obtained for cell hydraulic conductivity, Lp_c, and H⁺-ATPase activity, large reductions in the presence at NaCl or KCl and an ameliorative effect of Ca²⁺. However, fusicoccin (an activator of H⁺-ATPase) did not alter osmotic water permeability of protoplasts isolated from roots. Addition of Hg²⁺ inhibited both ATPase and aquaporins, but ATPase also contains Hg-binding sites. Therefore, the results indicate that H⁺-ATPase and aquaporin activities may not be related in pepper plants.     

Characterization of Na+ exclusion mechanisms of salt-tolerant reed plants in comparison with salt-sensitive rice plants

Salt-tolerant reed plants ( Phragmites communis Trinius) and salt-sensitive rice plants ( Oryza sativa L. cv. Kinmaze) were grown in salinized nutrient solutions up to 50 m M NaCl, and growth, Na⁺ contents and kinetics of ²²Na⁺ uptake and translocation were compared between the species to characterize the salt tolerance mechanisms operating in reed plants. When both plants were grown under the same salinity, Na⁺ contents of the shoots were lower in reed plants, although those of the roots were quite similar. The shoot base region of both species accumulated Na⁺ more than the leaf blades did. Sodium-22 uptake and pulse-chase experiments suggested that the lower Na⁺ transport rate from root to shoot could limit excessive Na⁺ accumulation in the reed shoot. There was a possibility that the apparently lower ²²Na⁺ transport rate to the shoot of reed plants was due to net downward Na⁺ transport from shoot base to root.     

Effects of phloridzin, metavanadate and oligomycin on membrane bound (Na++ K++ Mg2+)ATPase activity in sugar beet roots

To clarify the reaction mechanism of a (Na⁺+ K⁺+ Mg²⁺)ATPase activity in sugar beet roots ( Beta vulgaris L. cv. Monohill) phloridzin, oligomycin (inhibitors of animal ATPases) and metavanadate (NH₄VO₃) have been used. Kinetic studies showed that: 1) Phloridzin inhibition is uncompetitive with respect to MgATP and not influenced by Na⁺ or K⁺. 2) This inhibition is only found in preparations made in the absence of sucrose. 3) Oligomycin and vanadate inhibit the ATPase in different ways. Omission of sucrose from the preparation medium favours vanadate inhibition but suppresses oligomycin inhibition. 4) The kinetic pattern of the Na⁺ activation of the ATPase differs in preparations made in the absence and presence of sucrose, but that of K⁺ activation is the same. – These results indicate that inclusion as against omission of sucrose from the preparation medium causes a conformational change of the membrane fragments/vesicles, which then expose different surfaces to the surrounding medium.     

Effects of salt stress on H+- ATPase and H+-PPase activities of tonoplast-enriched vesicles isolated from sunflower roots

The control of ion concentration in the cytosol and the accumulation of ions in vacuoles are thought to be key factors in salt tolerance. These processes depend on the establishment in vacuolar membranes of an electrochemical H⁺ gradient generated by two distinct H⁺-translocating enzymes: a H⁺-PPase and a H⁺-ATPase. H⁺-lrans locating activities were characterized in tonoplast-enriched membrane fractions isolated by sucrose gradient centrifugation from sunflower ( Helianthus annuus L.) roots exposed for 3 days to different NaCl regimes. The 15/32% sucrose interface was enriched in membrane vesicles possessing a vacuolar-type H⁺-ATPase and a H⁺-PPase, as indicated by inhibitor sensitivity, pH optimum, substrate specificity, ion effects kinetic data and immunolabelling with specific antibodies. Mild and severe stress did not alter the pH profile, ion dependence, apparent K_m nor the amount of antigenic protein of either enzyme. Saline treatments slightly increased K⁺-stimulaied PPase activity with no change in ATPase activity, while both PP_i-dependent and NO₃-sensitive ATP-dependent H⁺ transport activities were strongly stimulated. These results are discussed in terms of an adaptative mechanism of the moderately tolerant sunflower plants to salt stress.     

Cation/proton antiporter complements of bacteria: why so large and diverse?

Most bacterial genomes have five to nine distinct genes predicted to encode transporters that exchange cytoplasmic Na⁺ and/or K⁺ for H⁺ from outside the cell, i.e. monovalent cation/proton antiporters. By contrast, pathogens that live primarily inside host cells usually possess zero to one such antiporter while other stress-exposed bacteria exhibit even higher numbers. The monovalent cation/proton antiporters encoded by these diverse genes fall into at least eight different transporter protein families based on sequence similarity. They enable bacteria to meet challenges of high or fluctuating pH, salt, temperature or osmolarity, but we lack explanations for why so many antiporters are needed and for the value added by specific antiporter types in specific settings. In this issue of Molecular Microbiology, analyses of the pH dependence of cytoplasmic [Na⁺], [K⁺], pH and transmembrane electrical potential in the 'poly extremophile' Natranaerobius thermophilus are the context for assessment of the catalytic properties of 12 predicted monovalent cation/proton antiporters in the genome of this thermophilic haloalkaliphile. The results provide a profile of adaptations of the poly extremophilic anaerobe, including a proposed role of cytoplasmic buffering capacity. They also provide new perspectives on two large monovalent cation/proton antiporter families, the NhaC and the cation/proton antiporter-3 antiporter families.     

The halophilic alkalithermophile Natranaerobius thermophilus adapts to multiple environmental extremes using a large repertoire of Na+(K+)/H+ antiporters

Natranaerobius thermophilus is an unusual extremophile because it is halophilic, alkaliphilic and thermophilic, growing optimally at 3.5 M Na⁺, pH^55°C 9.5 and 53°C. Mechanisms enabling this tripartite lifestyle are essential for understanding how microorganisms grow under inhospitable conditions, but remain unknown, particularly in extremophiles growing under multiple extremes. We report on the response of N. thermophilus to external pH at high salt and elevated temperature and identify mechanisms responsible for this adaptation. N. thermophilus exhibited cytoplasm acidification, maintaining an unanticipated transmembrane pH gradient of 1 unit over the entire extracellular pH range for growth. N. thermophilus uses two distinct mechanisms for cytoplasm acidification. At extracellular pH values at and below the optimum, N. thermophilus utilizes at least eight electrogenic Na⁺(K⁺)/H⁺ antiporters for cytoplasm acidification. Characterization of these antiporters in antiporter-deficient Escherichia coli KNabc showed overlapping pH profiles (pH 7.8–10.0) and Na⁺ concentrations for activity ( K _0.5 values 1.0–4.4 mM), properties that correlate with intracellular conditions of N. thermophilus . As the extracellular pH increases beyond the optimum, electrogenic antiport activity ceases, and cytoplasm acidification is achieved by energy-independent physiochemical effects (cytoplasmic buffering) potentially mediated by an acidic proteome. The combination of these strategies allows N. thermophilus to grow over a range of extracellular pH and Na⁺ concentrations and protect biomolecules under multiple extreme conditions.     

Overexpression of RCI2A decreases Na+ uptake and mitigates salinity-induced damages in Arabidopsis thaliana plants

We have investigated whether the overexpression of RCI2A gene causes an enhanced salt-tolerant phenotype in Arabidopsis thaliana . Although the growth of RCI2A -overexpressing transgenic plants was comparable with that of wild type under normal conditions, high salinity treatment caused decreased accumulation of Na⁺ and ameliorated suppression of the shoot growth of transgenic plants than that of wild type. Under high salinity treatment, the chlorophyll content of the shoots of wild-type plants significantly decreased compared with transgenic plants. The increases of malondialdehyde (MDA) and of H₂O₂ production caused by high salinity were greater in the shoots of wild type than in that of transgenic plants. These results suggest that overexpression of RCI2A can alleviate salinity-induced growth suppression and photooxidative damages via reducing Na⁺ uptake into the shoots.     

Puccinellia tenuiflora maintains a low Na+ level under salinity by limiting unidirectional Na+ influx resulting in a high selectivity for K+ over Na+

Puccinellia tenuiflora is a useful monocotyledonous halophyte that might be used for improving salt tolerance of cereals. This current work has shown that P. tenuiflora has stronger selectivity for K⁺ over Na⁺ allowing it to maintain significantly lower tissue Na⁺ and higher K⁺ concentration than that of wheat under short- or long-term NaCl treatments. To assess the relative contribution of Na⁺ efflux and influx to net Na⁺ accumulation, unidirectional ²²Na⁺ fluxes in roots were carried out. It was firstly found that unidirectional ²²Na⁺ influx into root of P. tenuiflora was significantly lower (by 31–37%) than in wheat under 100 and 150 m m NaCl. P. tenuiflora had lower unidirectional Na⁺ efflux than wheat; the ratio of efflux to influx was similar between the two species. Leaf secretion of P. tenuiflora was also estimated, and found the loss of Na⁺ content from leaves to account for only 0.0006% of the whole plant Na⁺ content over 33 d of NaCl treatments. Therefore, it is proposed that neither unidirectional Na⁺ efflux of roots nor salt secretion by leaves, but restricting unidirectional Na⁺ influx into roots with a strong selectivity for K⁺ over Na⁺ seems likely to contribute to the salt tolerance of P. tenuiflora .     

Effects of Cd2+ and EDTA on young sugar beets (Beta vulgaris). I. Cd2+ uptake and sugar accumulation

Sugar beets ( Beta vulgaris L. cv. Monohill) grown in a complete nutrient solution, were treated with Cd²⁺ (5 or 50 μ M ) and/or EDTA (10 or 100 μ M ) in different combinations. The Cd contents of five-week-old roots and shoots were determined by atomic absorption spectrophotometry, and the sucrose, glucose and fructose contents were measured enzymatically. The Cd²⁺ uptake in both roots and shoots shows a linear relationship to the concentration of free Cd²⁺ in the nutrient solution. This uptake is diminished in the presence of EDTA, suggesting that the Cd-EDTA complex is unable to penetrate the membranes. The contents of glucose, fructose and sucrose in both roots and shoots decrease with increasing uptake of free Cd²⁺. This may be a secondary effect caused by the inhibition of photosynthesis in the presence of Cd²⁺. EDTA reduces the inhibition of Cd²⁺ on sugar formation and accumulation. In the presence of EDTA alone the sugar content increases somewhat. EDTA slightly influences the dry weights of whole plants. The ratio roots:whole plants increases. Cd²⁺ (≤ 50 μ M ) increases the dry matter portion of roots by ca 30%, but not that of shoots.     

Characterization of membrane-bound MgATPases, purified by aqueous two-phase partitioning, from young sugar beet roots

Membrane-bound MgATPase activity from roots of young sugar beet ( Beta vulgaris L. cv. Monohill) was investigated in a membrane fraction purified by partition in an aqueous polymer two-phase system. After two steps of "washing" with fresh bottom phase (rich in dextran), the polyethylene glycol rich top phase (U₃) was practically free of mitochondrial membranes (cytochrome oxidase), and the remaining MgATPase activity showed high substrate specificity for ATP. An optimum for the MgATPase activity was found at pH 7. The activation by Na⁺ or K⁺ was strongest on the acid side without any observable shift in pH optimum. Oligomycin had no effect, but vanadate strongly inhibited the U₃ MgATPase and the K⁺ activation was lost. The complex activation pattern achieved by varying the Na⁺/K⁺ ratio at constant total concentration was interpreted as a synergistic (Na⁺+ K⁺)-activation. The U₃ fraction MgATP-ase activity showed a 4-fold increase in the presence of 0.01% Triton X-100 implying that the MgATPase activity is located in vesicles of which 75% or more are sealed with the ATP binding site on the inside. Comparison with the properties of plasma membrane. ATPases from other plants indicated that the U₃ fraction MgATPase was mainly of plasma membrane origin.