Comparison of the effects of salt-stress and alkali-stress on photosynthesis and energy storage of an alkali-resistant halophyte <Emphasis Type="Italic">Chloris virgata</Emphasis>

Seedlings of Chloris virgata were treated with varying (0–160 mM) salt-stress (SS; 1: 1 molar ratio of NaCl to Na₂SO₄) or alkali-stress (AS; 1: 1 molar ratio of NaHCO₃ to Na₂CO₃). To compare these effects, relative growth rates (RGR), stored energy, photosynthetic pigment contents, net photosynthetic rates, stomatal conductance, and transpiration rates were determined. Both stresses did not change significantly the photosynthetic parameters of C. virgata under moderate stress (below 120 mM). Photosynthetic ability decreased significantly only at high stress (160 mM). Thus C. virgata, a natural alkali-resistant halophyte, adapts better to both kinds of stress. The inhibition effects of AS on RGR and energy storage of C. virgata were significantly greater than that of SS of the same intensity. The energy consumption of C. virgata was considerably greater while resisting AS than while resisting SS.     

Enhancement of the salt tolerance of Triticum turgidum L. by the Kna1 locus transferred from the Triticum aestivum L. chromosome 4D by homoeologous recombination

Durum wheat, Triticum turgidum L. (2n= 4x=28, genome formula AABB) is inferior to bread wheat, T. aestivum L. (2n=6x=42, genome formula AABBDD), in the ability to exclude Na⁺ under salt strees, in the ratio of the accumulated K⁺ to Na⁺ in the leaves under salt stress, and in tolerance of salt stress. Previous work showed that chromosome 4D has a major effect on Na⁺ and K⁺ accumulation in the leaves of bread wheat. The 4D chromosome was recombined with chromosome 4B in the genetic background of durum wheat. The recombinants showed that Na⁺ exclusion and enhanced K⁺/Na⁺ ratio in the shoots were controlled by a single locus, Kna1, in the long arm of chromosome 4D. The recombinant families were grown in the field under non-saline conditions and two levels of salinity to determine whether Kna1 confers salt tolerance. Under salt stress, the Kna1 families had higher K⁺/Na⁺ ratios in the flag leaves and higher yields of grain and biomass than the Kna1 ^- families and the parental cultivars. Kna1 is, therefore, one of the factors responsible for the higher salt tolerance of bread wheat relative to durum wheat. The present work provides conceptual evidence that tolerance of salt stress can be transferred between species in the tribe Triticeae.     

NhaK,a novel monovalent cation/H<Superscript>+</Superscript> antiporter of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Four Na⁺/H⁺ antiporters, Mrp, TetA(L), NhaC, and MleN have so far been described in Bacillus subtilis 168. We identified an additional Na⁺/H⁺ antiporter, YvgP, from B. subtilis that exhibits homology to the cation: proton antiporter-1 (CPA-1) family. The yvgP-dependent complementation observed in a Na⁺(Ca²⁺)/H⁺ antiporter-defective Escherichia coli mutant (KNabc) suggested that YvgP effluxed Na⁺ and Li⁺. In addition, effects of yvgP expression on a K⁺ uptake-defective mutant of E. coli indicated that YvgP also supported K⁺ efflux. In a fluorescence-based assay of everted membrane vesicles prepared from E. coli KNabc transformants, YvgP-dependent Na⁺ (K⁺, Li⁺, Rb⁺)/H⁺ antiport activity was demonstrated. Na⁺ (K⁺, Li⁺)/H⁺ activity was higher at pH 8.5 than at pH 7.5. Mg²⁺, Ca²⁺ and Mn²⁺ did not serve as substrates but they inhibited YvgP antiport activities. Studies of yvgP expression in B. subtilis, using a reporter gene fusion, showed a significant constitutive level of expression that was highest in stationary phase, increasing as stationary phase progressed. In addition, the expression level was significantly increased in the presence of added K⁺ and Na⁺.     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane     

Fluorescent labeling of (Na + K)-ATPase by 5-iodoacetamidofluorescein

5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na⁺ + K⁺)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K⁺-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na⁺, K⁺, Mg²⁺, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na⁺, and Mg²⁺, and decreased by K⁺. These fluorescence changes likely reflect ligand-induced stabilization of the E₁ or E₂ states of the enzyme.     

Cloning,sequencing and expression of the nhaA and nhaR genes from Salmonella entiritidis

Na⁺/H⁺ antiporter activity is wide-spread and plays essential physiological roles. We found that several Enterobacteriaceae share conserved sequences with nhaA, the gene coding for an E. coli antiporter. A nhaA strain which is sensitive to Na⁺ and Li⁺, was used to clone by complementation a DNA fragment from Salmonella enteritidis which confers resistance to the ions. The cloned fragment increased Na⁺/H⁺ antiport activity in membranes isolated from strains carrying the respective hybrid plasmid. DNA sequence analysis of the insert revealed two open reading frames. Both encode putative polypeptides which are closely homologous to the nhaA and nhaR gene products from Escherichia coli. The antiporter activity displays properties very similar to that of the E. coli NhaA, namely, it is activiated by alkaline pH and recognizes Li⁺ with high affinity.Abbreviations _H ⁺ Proton electrochemical potential - pH transmembrane pH gradient - _Na ⁺ Sodium electrochemical potential - SDS Sodium dodecyl sulfate - CIP Calf intestine alkaline phosphates - ORF open reading frame     

Over-expression of a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene improves salt tolerance in an upland rice

To develop a salt-tolerant upland rice cultivar (Oryza sativa L.), OsNHX1, a vacuolar-type Na⁺/H⁺ antiporter gene from rice was transferred into the genome of an upland rice cultivar (IRAT109), using an Agrobacterium-mediated method. Seven independent transgenic calli lines were identified by polymerase chain reaction (PCR) analysis. These 35S::OsNHX1 transgenic plants displayed a little accelerated growth during seedling stage but showed delayed flowering time and a slight growth retardation phenotype during late vegetative stage, suggesting that the OsNHX1 has a novel function in plant development. Northern and western blot analyses showed that the expression levels of OsNHX1 mRNA and protein in the leaves of three independent transgenic plant lines were significantly higher than in the leaves of wild type (WT) plants. T₂ generation plants exhibited increased salt tolerance, showing delayed appearance and development of damage or death caused by salt stress, as well as improved recovery upon removal from this condition. Several physiological traits, such as increased Na⁺ content, and decreased osmotic potential in transgenic plants grown in high saline concentrations, further indicated that the transgenic plants had enhanced salt tolerance. Our results suggest the potential use of these transgenic plants for further agricultural applications in saline soil.     

Mathematical modelling of ATP, K and Na interactions with (Na + K)-ATPase occurring under equilibrium conditions

The controlling effect of ATP, K⁺ and Na⁺ on the rate of (Na⁺ + K⁺)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions.

1. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper.

2. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K⁺ and Na⁺ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits.

3. 3. The apparent dissociation constants for ATP of the enzyme-ATP complex or of the enzyme-(ATP)₂ complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K⁺ and Na⁺ were absent or K⁺ and K⁺ plus Na⁺, respectively, were present in the experiments.

4. 4. The origin of the high and the low affinity site for binding of ATP to the (Na⁺ + K⁺)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-Cl, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry.

RETRACTED ARTICLE: Analysis of the physiological mechanism of salt-tolerant transgenic rice carrying a vacuolar Na+/H+ antiporter gene from Suaeda salsa

Salt stress is one of the most serious factors limiting the productivity of agricultural crops. Increasing evidence has demonstrated that vacuolar Na⁺/H⁺ antiporters play a crucial role in plant salt tolerance. In the present study, we expressed the Suaeda salsa vacuolar Na⁺/H⁺ antiporter SsNHX1 in transgenic rice to investigate whether this can increase the salt tolerance of rice, and to study how overexpression of this gene affected other salt-tolerant mechanisms. It was found that transgenic rice plants showed markedly enhanced tolerance to salt stress and to water deprivation compared with non-transgenic controls upon salt stress imposition under outdoor conditions. Measurements of ion levels indicated that K⁺, Ca²⁺ and Mg²⁺ contents were all higher in transgenic plants than in non-transformed controls. Furthermore, shoot V-ATPase hydrolytic activity was dramatically increased in transgenics compared to that of non-transformed controls under salt stress conditions. Physiological analysis also showed that the photosynthetic activity of the transformed plants was higher whereas the same plants had reduced reactive oxygen species generation. In addition, the soluble sugar content increased in the transgenics compared with that in non-transgenics. These results imply that up-regulation of a vacuolar Na⁺/H⁺ antiporter gene in transgenic rice might cause pleiotropic up-regulation of other salt-resistance-related mechanisms to improve salt tolerance.Fengyun Zhao and Zenglan Wang contributed equally to this work.     

Salt tolerance conferred by overexpression of Arabidopsis vacuolar Na+/H+ antiporter gene AtNHX1 in common buckwheat (Fagopyrum esculentum)

Agriculture productivity is severely affected by soil salinity. One possible mechanism by which plants could survive salt stress is to compartmentalize sodium ions away from the cytosol. In the present work, transgenic buckwheat plants overexpressing AtNHX1, a vacuolar Na⁺/H⁺ antiporter gene from Arabidopsis thaliana, were regenerated after transformation with Agrobacterium tumefaciens. These plants were able to grow, flower and accumulate more rutin in the presence of 200 mmol/l sodium chloride. Moreover, the content of important nutrients in buckwheat was not affected by the high salinity of the soil. These results demonstrated the potential value of these transgenic plants for agriculture use in saline soil.     

Comparison of effects of salt and alkali stresses on the growth and photosynthesis of wheat

The seedlings of wheat were treated by salt-stress (SS, molar ratio of NaCl: Na₂SO₄ = 1: 1) and alkali-stress (AS, molar ratio of NaHCO₃: Na₂CO₃ = 1: 1). Relative growth rate (RGR), leaf area, and water content decreased with increasing salinity, and the extents of the reduction under AS were greater than those under SS. The contents of photosynthetic pigments did not decrease under SS, but increased at low salinity. On the contrary, the contents of photosynthetic pigments decreased sharply under AS with increasing salinity. Under SS, the changes of net photosynthetic rate (P _N), stomatal conductance (g _s), and transpiration rate (E) were similar and all varied in a single-peak curve with increasing salinity, and they were lower than those of control only at salinity over 150 mM. Under AS, P _N, g _s, and E decreased sharply with rising salinity. The decrease of g _s might cause the obvious decreases of E and intercellular CO₂ concentration, and the increase of water use efficiency under both stresses. The Na⁺ content and Na⁺/K⁺ ratio in shoot increased and the K⁺ content in shoot decreased under both stresses, and the changing extents under AS were greater than those under SS. Thus SS and AS are two distinctive stresses with different characters; the destructive effects of AS on the growth and photosynthesis of wheat are more severe than those under SS. High pH is the key feature of the AS that is different from SS. The buffer capacity is essentially the measure of high pH action on plant. The deposition of mineral elements and the intracellular unbalance of Na⁺ and K⁺ caused by the high pH at AS might be the reason of the decrease of P _N and g _s and of the destruction of photosynthetic pigments.     

Cloning and functional expression in <Emphasis Type="Italic">Saccharomyces cereviae</Emphasis> of a K<Superscript>+</Superscript> transporter,AlHAK, from the graminaceous halophyte, <Emphasis Type="Italic">Aeluropus littoralis</Emphasis>

High-affinity K⁺ transporters play an important role in K⁺ absorption of plants. We isolated a HAK gene from Aeluropus littoralis, a graminaceous halophyte. The amino acid sequence of AlHAK showed high homology with HAK transporters obtained from Oryza sativa (82%) and Hordeum vulgare (82%). When expressed in Saccharomyces cereviae WΔ3, AlHAK performed high-affinity K⁺ uptake with a K_m value of 8 μM, and the growth of transformants was dramatically inhibited by 150 mM Rb⁺ and 150 mM Cs⁺ but less affected by 300 mM Na⁺. AlHAK may thus improve the capacity of plants to maintain a high cytosolic K⁺/Na⁺ ratio at high salinity.     

Molecular Cloning and Functional Analysis of a Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Gene <Emphasis Type="Italic">ThNHX1</Emphasis> from a Halophytic Plant <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Na⁺/H⁺ exchanger catalyzes the countertransport of Na⁺ and H⁺ across membranes. Using the rapid amplification of cDNA ends method, a Na⁺/H⁺ antiporter gene (ThNHX1) was isolated from a halophytic plant, salt cress (Thellungiella halophila). The deduced amino acid sequence contained 545 amino acid residues with a conserved amiloride-binding domain (⁸⁷LFFIYLLPPI⁹⁶) and shared more than 94% identity with that of AtNHX1 from Arabidopsis thaliana. The ThNHX1 mRNA level was upregulated by salt and other stresses (abscisic acid, polyethylene glycol, and high temperature). This gene partially complemented the Na⁺/Li⁺-sensitive phenotype of a yeast mutant that was deficient in the endosomal–vacuolar Na⁺/H⁺ antiporter ScNHX1. Overexpression of ThNHX1 in Arabidopsis increased salt tolerance of transgenic plants compared with the wild-type plants. In addition, the silencing of ThNHX1 gene in T. halophila caused the transgenic plants to be more salt and osmotic sensitive than wild-type plant. Together, these results suggest that ThNHX1 may function as a tonoplast Na⁺/H⁺ antiporter and play an important role in salt tolerance of T. halophila. Chunxia Wu, Xiuhua Gao, and Xiangqiang Kong contributed equally to this work.     

短期NaCl胁迫对不同小麦品种幼苗K+吸收和Na+、K+积累的影响

为研究盐胁迫下小麦幼苗生长及Na⁺、K⁺的吸收和积累规律,以中国春、洲元9369和长武134等3种耐盐性不同小麦品种为材料,采用非损伤微测技术检测盐胁迫2 d后的根系K⁺离子流变化,并对植株体内的Na⁺、K⁺含量进行测定。结果表明:短期(2d)盐胁迫对小麦生长有抑制作用,且对根系的抑制大于地上部,耐盐品种下降幅度小于盐敏感品种。盐胁迫下,小麦根际的 K⁺大量外流,盐敏感品种中国春K⁺流速显著高于耐盐品种长武134,最高可达15倍。小麦幼苗地上部分和根系均表现为Na⁺积累增加,K⁺积累减少,Na⁺/K⁺比随盐浓度增加而上升。中国春限Na⁺能力显著低于长武134,Na⁺/K⁺则显著高于长武134。综上所述,盐胁迫下造成小麦组织器官中Na⁺/K⁺比上升的主要原因是根系K⁺大量外流和Na⁺的过量积累,耐盐性不同的小麦品种间差异显著,并认为根系对K⁺的保有能力可能是作物耐盐性评价的一个重要指标。     

Furosemide-sensitive K+ (Rb+) transport in human erythrocytes: Modes of operation,dependence on extracellular and intracellular Na+, kinetics,pH dependency and the effect of cell volume and N-ethylmaleimide

Summary The effect of extracellular and intracellular Na⁺ (Na _o ⁺ , Na _i ⁺ ) on ouabain-resistant, furosemide-sensitive (FS) Rb⁺ transport was studied in human erythrocytes under varying experimental conditions. The results obtained are consistent with the view that a (1 Na⁺+1 K⁺+2 Cl^–) cotransport system operates in two different modes: modei) promoting bidirectional 11 (Na⁺–K⁺) cotransport, and modeii) a Na _o ⁺ -independent 11 K _o ⁺ /K _i ⁺ exchange requiring Na _i ⁺ which, however, is not extruded. The activities of the two modes of operation vary strictly in parallel to each other among erythrocytes of different donors and in cell fractions of individual donors separated according to density. Rb⁺ uptake through Rb _o ⁺ /K _i ⁺ exchange contributes about 25% to total Rb⁺ uptake in 145mm NaCl media containing 5mm RbCl at normal Na _i ⁺ (pH 7.4). Na⁺–K⁺ cotransport into the cells occurs largely additive to K⁺/K⁺ exchange. Inward Na⁺–Rb⁺ cotransport exhibits a substrate inhibition at high Rb _o ⁺ . With increasing pH, the maximum rate of cotransport is accelerated at the expense of K⁺/K⁺ exchange (apparent pK close to pH 7.4). The apparentK _m ^Rb _o ⁺ of Na⁺–K⁺ cotransport is low (2mm) and almost independent of pH, and high for K⁺/K⁺ exchange (10 to 15mm), the affinity increasing with pH. The two modes are discussed in terms of a partial reaction scheme of (1 Na⁺+1 K⁺+2 Cl^–) cotransport with ordered binding and debinding, exhibiting a glide symmetry (first on outside = first off inside) as proposed by McManus for duck erythrocytes (McManus, T.J., 1987,Fed. Proc., in press). N-ethylmaleimide (NEM) chemically induces a Cl^–-dependent K⁺ transport pathway that is independent of both Na _o ⁺ and Na _i ⁺ . This pathway differs in many properties from the basal, Na _o ⁺ -independent K⁺/K⁺ exchange active in untreated human erythrocytes at normal cell volume. Cell swelling accelerates a Na _o ⁺ -independent FS K⁺ transport pathway which most probably is not identical to basal K⁺/K⁺ exchange. K _o ⁺ o ⁺

o ⁺ o ²⁺ reduce furosemide-resistant Rb⁺ inward leakage relative to choline _o ⁺ .     

The efficiency of (Na + K)-ATPase in tumorigenic cells

The efficiency of (Na⁺ + K⁺)-ATPase (i.e. the amount of K⁺ pumped per ATP hydrolyzed) in intact tumorigenic cells was estimated in this study. This was accomplished by simultaneously measuring the rate of ouabain-sensitive K⁺ uptake and oxygen consumption in tumorigenic cell suspensions during the reintroduction of K⁺ to K⁺-depleted cells. The ATP turnover was then estimated by assuming 5.6–6 ATP/O₂ as the stoichiometry of NADH-linked respiration in these cells. In the three cell lines tested (hamster and chick embryo cells transformed with Rous sarcoma virus and Ehrlich ascites cells), the K⁺/ATP ratio was approximately 2, the same value as that found in normal tissues. Furthermore, only 20% of the total ATP production of these cells was used by (Na⁺ + K⁺)-ATPase.     

长苞香蒲对人工盐碱湿地Na+和K+的吸收与转运特征

为揭示长苞香蒲(Typha domingensis)对盐生湿地生态系统中Na⁺和K⁺的吸收与转运特征,探讨长苞香蒲对盐生湿地的生态修复效果,该研究采用人工模拟盐生湿地的方法,设置CK(对照)、T1(浇灌100 mmol·L^-1盐水)、T2(浇灌200 mmol·L^-1盐水)及T3(浇灌300 mmol·L^-1盐水)4种不同盐浓度的人工湿地生态系统,并分别于5月5日(开始盐胁迫处理,S0)、5月30日(S1)、6月30日(S2)和7月30日(S3)测量其株高和干重、植株地上与地下部分Na⁺和K⁺的含量以及底泥和水体中Na⁺和K⁺的含量以分析长苞香蒲对盐碱湿地的脱盐作用。结果表明：(1)各处理的长苞香蒲的株高和干重随着处理时间的延长呈增加趋势,但与CK相比,各处理生长量随盐浓度升高出现下降趋势。(2)高浓度盐处理(T3)使长苞香蒲的地上部分和地下部分的Na⁺分别增加了2.5...     

Overexpression of AeNHX1, a root-specific vacuolar Na+/H+ antiporter from Agropyron elongatum, confers salt tolerance to Arabidopsis and Festuca plants

Agropyron elongatum, a species in grass family, has a strong tolerance to salt stress. To study the molecular mechanism of Agropyron elongatum in salt tolerance, we isolated a homolog of Na⁺/H⁺ antiporters from the root tissues of Agropyron plants. Sequence analysis revealed that this gene encodes a putative vacuolar Na⁺/H⁺ antiporter and was designated as AeNHX1. The AeNHX1–GFP fusion protein was clearly targeted to the vacuolar membrane in a transient transfection assay. Northern analysis indicated that AeNHX1 was expressed in a root-specific manner. Expression of AeNHX1 in yeast Na⁺/H⁺ antiporter mutants showed function complementation. Further, overexpression of AeNHX1 promoted salt tolerance of Arabidopsis plants, and improved osmotic adjustment and photosynthesis which might be responsible for normal development of transgenic plants under salt stress. Similarly, AeNHX1 also functioned in transgenic Festuca plants. The results suggest that this gene might function in the roots of Agropyron plants, and its expression is involved in the improvement of salt tolerance.