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Overexpression of salt-tolerant glutaminase from Micrococcus luteus K-3 in Escherichia coli and its purification.

A high-expression plasmid, pKSGHE3-1, containing the salt-tolerant glutaminase (EC 3.5.1.2) from marine bacterium Micrococcus luteus K-3 was constructed. pKSGHE3-1 was made by inserting the DNA fragment (1.43 kb) containing the structural gene synthesized by polymerase chain reaction into the downstream region of the tac promoter of expression vector pKK223-3. The translational start codon was located 10 bases downstream of the Shine-Dalgarno sequence (AGGA) of pKK223-3. Escherichia coli JM109 transformed with pKSGHE3-1 exhibited more than 190-fold higher glutaminase activity than M. luteus K-3 under optimal culture conditions. The enzyme was purified to homogeneity through three column chromatography steps with a final yield of 17.1%. The recombinant enzyme showed the same enzymatic properties, including salt tolerance, as those of M. luteus K-3. This glutaminase expression system allows the production of sufficient quantities of glutaminase for basic structure-function studies including chemical modification and future X-ray crystallization analysis.

Bacterial Proteins↗

Overexpression of NtHAL3 genes confers increased levels of proline biosynthesis and the enhancement of salt tolerance in cultured tobacco cells.

The Hal3 protein of Saccharomyces cerevisiae inhibits the activity of PPZ1 type-1 protein phosphatases and functions as a regulator of salt tolerance and cell cycle control. In plants, two HAL3 homologue genes in Arabidopsis thaliana, AtHAL3a and AtHAl3b, have been isolated and the function of AtHAL3a has been investigated through the use of transgenic plants. Expressions of both AtHAL3 genes are induced by salt stress. AtHAL3a overexpressing transgenic plants exhibit improved salt and sorbitol tolerance. In vitro studies have demonstrated that AtHAL3 protein possessed 4'-phosphopantothenoylcysteine decarboxylase activity. This result suggests that the molecular function of plant HAL3 genes is different from that of yeast HAL3. To understand the function of plant HAL3 genes in salt tolerance more clearly, three tobacco HAL3 genes, NtHAL3a, NtHAL3b, and NtHAL3c, from Nicotiana tabacum were identified. NtHAL3 genes were constitutively expressed in all organs and under all conditions of stress examined. Overexpression of NtHAL3a improved salt, osmotic, and lithium tolerance in cultured tobacco cells. NtHAL3 genes could complement the temperature-sensitive mutation in the E. coli dfp gene encoding 4'-phosphopantothenoyl-cysteine decarboxylase in the coenzyme A biosynthetic pathway. Cells overexpressing NtHAL3a had an increased intracellular ratio of proline. Taken together, these results suggest that NtHAL3 proteins are involved in the coenzyme A biosynthetic pathway in tobacco cells.

Amino Acid Sequence↗

Expression of abscisic acid-responsive element-binding protein in salt-tolerant indica rice (Oryza sativa L. cv. Pokkali).

As the products of abiotic stress and ABA inducible genes are predicted to play an important role in the mechanism of salt tolerance, the expression of transcription factor that recognizes abscisic acid-responsive element (ABRE) is likely to be regulated when plants are exposed to abiotic stress. Northern analysis of total RNA from control and salt-treated 10-day-old Pokkali (salt tolerant) rice plants was performed to find out the level of transcripts homologous to wheat cDNA (GC19) for EmBP-1 (bZIP class factor), a transcription factor that recognizes ABRE. Salinity stress (72 h)-induced accumulation of two transcripts, of 2.0 kb (r2.0) and 1.5 kb (r1.5), in roots was detected. Both transcripts were detectable even after 6 h of salt or abscisic acid treatment, whereas sheath and lamina showed constitutive levels of r1.5 transcript. When 32P-labeled DNA containing ABRE was used in a gel mobility shift assay, a low level of complex formation by binding factor was detected from the nuclear extract of lamina of control rice plants. Quantitative enhancement of complex formation was found with the nuclear extract prepared from the lamina of plants treated with 200 mM NaCl for 26 h over control nuclear extract, suggesting a step of regulation of expression of ABRE-binding protein in response to salinity stress. South-western blot analysis of equal amounts of nuclear proteins of lamina showed binding of 32P-labeled ABRE-DNA with two polypeptides (22-28 kDa) present at constitutive levels in control or NaCl-treated plants. Preincubation of the laminar nuclear extract of control plants, with spermidine or proline at 5 mM concentration showed quantitative enhancement of ABRE binding activity. Kinetics of spermidine stimulation showed gradual increase of complex formation from 5 mM concentration. Similarly, addition of GTP to the control nuclear extract also showed quantitative enhancement of complex formation and heparin was found to inhibit GTP activated complex formation by about 25%. Results may suggest the presence of ABRE binding protein in presynthesized and inactive form in control plants and GTP mediated activation is probably one of the way to regulate the expression of ABRE-binding factor.

Abscisic Acid↗

Pretreatment of seed with H2O2 improves salt tolerance of wheat seedlings by alleviation of oxidative damage and expression of stress proteins.

Increased salinity is a stringent problem to crop production while seed pretreatment can effectively induce salt tolerance in plants. Hydrogen peroxide (H(2)O(2)), a stress signal molecule, was evaluated as seed treatment to produce the metabolic changes, which could lead to improved salt tolerance in wheat. Soaking in 1, 40, 80 and 120 microM H(2)O(2) revealed a low penetration, reaching maximum at 5h (2.58+/-0.23 micro mol g(-1) fresh seeds at 120 microM) and declining thereafter to the level of water control by 8h. This revealed the activation of antioxidants and H(2)O(2) scavenging in seed after 5h. Seeds treated with 1-120 microM H(2)O(2) for 8h and germinated in saline (150 mM NaCl) medium curtailed the mean germination time (MGT) being even less than water controls. Level of H(2)O(2) in seedlings arising from H(2)O(2)-treated seeds grown under salinity was markedly lower than salinized controls, suggesting the operation of antioxidant system in them. These seedlings exhibited better photosynthetic capacity, particularly the stomatal conductance (gs), thus improving the leaf gas exchange due to stomatal component of photosynthesis. Moreover, H(2)O(2) treatment improved leaf water relations and maintained turgor. Although Na(+) and Cl(-) content increased due to salinity, H(2)O(2)-treated seedlings displayed greater tissue K(+), Ca(2+), NO(3)(-) PO(4)(3-) levels and improved K(+):Na(+) ratio. H(2)O(2) treatment enhanced the membrane properties, as revealed from greatly reduced relative membrane permeability (RMP) and less altered ion leakage pattern (comparable to water controls). Seedlings exhibited the expression of two heat-stable (stress) proteins with apparent molecular masses of 32 and 52 kDa. Results suggest that H(2)O(2) signals the activation of antioxidants in seed, which persists in the seedlings to offset the ion-induced oxidative damage. These changes led to the expression of stress proteins and improved physiological attributes, which supported the seedling growth under salinity.

Adaptation, Physiological↗

Characterization of a salt-tolerant extracellular a-amylase from Bacillus dipsosauri.

AIMS: The aims of this study were to purify and characterize an extracellular alpha-amylase from the salt-tolerant bacterium Bacillus dipsosauri. METHODS AND RESULTS: An extracellular alpha-amylase from B. dipsosauri strain DD1 was studied using the synthetic substrate 2-chloro-4-nitrophenyl-alpha-D-maltotrioside. Formation of the enzyme was induced by starch, repressed by D-glucose and highest after growth in medium containing 1.0 mol l-1 KCl. The alpha-amylase activity increased with KCl concentration, showed a pH optimum of 6.5, was stable up to 60 degrees C and was stimulated by 1.0 mol l-1 Na2SO4. The enzyme was purified from spent culture medium to apparent homogeneity by precipitation with ethanol, ion-exchange chromatography on DEAE-cellulose, centrifugal membrane filtration and gel-filtration chromatography on BioGel P-100. The purified enzyme had a denatured molecular mass of about 80 kDa but behaved on non-denaturing polyacrylamide gels as if it had a mass of about 30 kDa. The enzyme was partially inhibited by glucose-containing oligosaccharides of increasing length and strongly inhibited by the divalent cations Cd2+ and Zn2+. CONCLUSIONS: The extracellular alpha-amylase from B. dipsosauri strain DD1 was purified to homogeneity and found to exhibit an unusually high degree of salt tolerance. SIGNIFICANCE AND IMPACT OF THE STUDY: The alpha-amylase from B. dipsosauri differs from previously described enzymes and may be useful for the processing of starches under high-salt conditions.

Aerobiosis↗

Salt tolerance of an aqueous solution of a novel amphiphilic polysaccharide derivative.

An aqueous solution of an amphiphilic polysaccharide derivative, hydrophobically (stearyl alkyl group) and hydrophilically (sulfonic-acid salt group) modified hydroxyethylcellulose (HHM-HEC), showed increased viscosity, elasticity, and thixotropic properties in response to the addition of monovalent and divalent salts. Furthermore, the HHM-HEC solution had a transparent appearance at a NaCl concentration of 7 wt %. Since it showed superior salt tolerance to HEC, we focused attention on the two substituents of HHM-HEC and prepared HEC derivatives, namely, hydrophobically modified hydroxyethylcellulose (R-HEC), hydrophilically modified hydroxyethylcellulose (S-HEC), and nonmodified hydroxyethylcellulose (HEC). In addition, we used oscillatory, thixotropic, and fluorometric methods to compare the rheological properties of HHM-HEC with those of other derivatives in the presence of NaCl and ZnCl2, and attempted to elucidate the respective roles of the two substituents of HHM-HEC solution in the salt-tolerance mechanism. As the NaCl concentration in the HHM-HEC solution increased, the values of the elastic modulus G' and the viscous modulus G'' increased, and, moreover, the relative intensities of the first (I1 = 372 nm) and the third (I3 = 383 nm) vibronic bands of the pyrene monomer emission spectrum (the I1/I3 ratio) decreased. These results suggested that the added salt strengthened the three-dimensional network structure of the HHM-HEC polymer by the formation of cross-linkages through the association of hydrophobic substituents. This hydrophobic substituent was therefore essential in allowing HHM-HEC to exhibit a high viscosity in a salt solution. Although the R-HEC solution showed a higher viscosity than did the HHM-HEC solution in the absence of added salts, it became cloudy and lost its viscosity at high NaCl concentrations, apparently because of the shrinkage of its network structure. This signified that the hydrophilic substituent was essential for the sufficient solubility of HHM-HEC to show its rheological properties in a salt-rich solution. We propose to explain how the viscosity of HHM-HEC increases in the presence of salts as follows: Added salts weaken the electrostatic repulsion between the hydrophilic substituents, thereby enhancing the interactions of hydrophobic substituents and consequently increasing the rigidity of the HHM-HEC solution.

Journal Article↗

Response of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii to salt-dependent oxidative stress: increased activities of antioxidant enzymes in root plastids.

Root plastids of the cultivated tomato Lycopersicon esculentum (Lem) exhibited salt-induced oxidative stress as indicated by the increased H2O2 and lipid peroxidation levels which were accompanied with increased contents of the oxidized forms of ascorbate and glutathione. In contrast, H2O2 level decreased, lipid peroxidation level slightly decreased and the levels of the reduced forms of ascorbate and glutathione increased in plastids of L. pennellii (Lpa) species in response to salinity. This better protection of Lpa root plastids from salt-induced oxidative stress was correlated with increased activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidases (POD), monodehydroascorbate reductase (MDHAR), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and phospholipid hydroperoxide glutathione peroxidase (PHGPX). In the plastids of both species, activities of SOD, APX, and POD could be resolved into several isozymes. In Lem plastids two Cu/ZnSOD isozymes were found whereas in Lpa an additional FeSOD type could also be detected. In response to salinity, activities of selected SOD, APX, and POD isozymes were increased in Lpa, while in Lem plastids the activities of most of SOD and POD isozymes decreased. Taken together, it is suggested that plastids play an important role in the adaptation of Lpa roots to salinity.

Ascorbate Peroxidases↗

Effects of Salinity on Stomatal Conductance, Photosynthetic Capacity, and Carbon Isotope Discrimination of Salt-Tolerant (Gossypium hirsutum L.) and Salt-Sensitive (Phaseolus vulgaris L.) C(3) Non-Halophytes.

The effects of salinity on growth, stomatal conductance, photosynthetic capacity, and carbon isotope discrimination (Delta) of Gossypium hirsutum L. and Phaseolus vulgaris L. were evaluated. Plants were grown at different NaCl concentrations from 10 days old until mature reproductive structures were formed. Plant growth and leaf area development were strongly reduced by salinity, in both cotton and bean. Stomatal conductance also was reduced by salinity. The Delta always declined with increasing external salinity concentration, indicating that stomatal limitation of photosynthesis was increased. In cotton plant dry matter, Delta correlated with the ratio of intercellular to atmospheric CO(2) partial pressures (p(l)/p(a)) calculated by gas exchange. This correlation was not clear in bean plants, although Delta showed a more pronounced salt induced decline in bean than in cotton. Possible effects of heterogeneity of stomatal aperture and consequent overestimation of p(l) as determined from gas exchange could explain these results. Significant differences of Delta between leaf and seed material were observed in cotton and bean. This suggests different patterns of carbon allocation between leaves and seeds. The photon yield of O(2) evolution determined at rate-limiting photosynthetic photon flux density was insensitive to salinity in both species analyzed. The light- and CO(2)-saturated rate of CO(2) uptake and O(2) evolution showed a salt induced decline in both species. Possible explanations of this observation are discussed. O(2) hypersensitivity was observed in salt stressed cotton plants. These results clearly demonstrate that the effect of salinity on assimilation rate was mostly due to the reduction of stomatal conductance, and that calculation of p(l) may be overestimated in salt stressed plants, because of heterogeneity of stomatal aperture over the leaf surface.

Journal Article↗

Growth of Escherichia coli in human urine: role of salt tolerance and accumulation of glycine betaine.

Glycine betaine is a powerful osmoprotectant molecule present in the inner medulla of the kidney and excreted into urine. It may be responsible for the ability of Escherichia coli to grow in hypertonic urine. Also, strains of E. coli that cause urinary tract infections may be more salt-tolerant than strains from other sites. To explore these questions, 301 isolates from blood, urine, or stool and 12 representative enteric strains were examined. Tolerance varied from 0.1 to 0.7 M NaCl (median, 0.5) in minimal medium. There were no significant differences in salt tolerance by site of isolation. A salt-sensitive enteric strain that responded poorly to glycine betaine and mutant strains lacking the ability to synthesize or transport glycine betaine did not grow well in hypertonic urine. Accumulation of glycine betaine appears to be a mechanism by which E. coli can adapt to external osmotic forces and grow in hypertonic urine.

Betaine↗

A procedure for enrichment and isolation of mutants of the salt-tolerant yeast Debaryomyces hansenii having altered glycerol metabolism.

The salt-tolerant yeast Debaryomyces hansenii produces and accumulates glycerol when subjected to salt stress, whereby the buoyant density of the cells is changed. This property allows for enrichment of mutants with altered glycerol metabolism by density gradient centrifugation. Colonies derived from cells with rapidly changing density following an osmotic shock were screened for increased glycerol production by observing their ability to support growth of a glycerol-requiring strain of Escherichia coli. The glycerol overproducting phenotype of two isolates was confirmed by chemical analysis.

Centrifugation, Density Gradient↗

Chloride absorption in salt-sensitive Carrizo citrange and salt-tolerant Cleopatra mandarin citrus rootstocks is linked to water use.

In this work, seedlings of two citrus rootstocks, the salt-tolerant Cleopatra mandarin (Citrus reshni Hort. ex Tan.) and the salt-sensitive Carrizo citrange (Citrus sinensis [L.] Osb. x Poncirus trifoliata [L.] Raf.) were used to study the relationship between chloride and water uptake. The results indicated that net chloride uptake rates in both genotypes were alike and decreased linearly with the time of salinity exposure, although they were more rapidly reduced in the tolerant genotype. In each rootstock, chloride uptake rates paralleled the decreases in transpiration rates. When transpiration was modified, concomitant changes in leaf Cl(-) concentrations were observed. There was a high positive correlation between total chloride content per plant and total water absorbed. In addition, the data indicate that the tolerant genotype "excluded" more chloride, i.e. it absorbed lower amounts of chloride per volume of water. Cleopatra also possessed a less efficient root system for water uptake and a higher shoot-to-root ratio. The results show that, overall, chloride absorption is linked to water use and that further tolerance in Cleopatra is mostly conferred by superior root resistance to Cl(-) uptake. Therefore, it is proposed that chloride absorption and, hence, salt tolerance in citrus depends to a great extent upon water use.

Adaptation, Physiological↗

Regulation of the potassium to sodium ratio and of the osmotic potential in relation to salt tolerance in yeasts.

By using the isotope pairs (22)Na-(24)Na and (42)K-(86)Rb, the uptake and retention of Na and K was studied in the salt-tolerant Debaryomyces hansenii and in the less tolerant Saccharomyces cerevisiae at NaCl levels of 4 mm and 0.68, 1.35, and 2.7 m in the medium. The ratio of K to Na is much higher in the cells than in the media, and higher in D. hansenii than in S. cerevisiae under comparable conditions. The difference between the two species is due to a better Na extrusion and a better uptake of K in D. hansenii. The kinetics of ion transport show that at about the time when extrusion of Na could be demonstrated in D. hansenii, K-Rb previously lost to an easily washable compartment of the cells was reabsorbed in both organisms. More H(+) was given off from S. cerevisiae than from D. hansenii in the course of these events. The findings fit the working hypothesis tested, which regards salt tolerance as partly dependent on the ability to mobilize energy to extrude Na from the cells and to take up K. The volume changes in S. cerevisiae are greater and are more slowly overcome than those in D. hansenii. The total salt level of the cells is not sufficient to counteract the osmotic potential of the medium, so that additional osmoregulatory mechanisms must be involved in determining halotolerance.

Hydrogen-Ion Concentration↗

Suppression of Arabidopsis vesicle-SNARE expression inhibited fusion of H2O2-containing vesicles with tonoplast and increased salt tolerance.

Intracellular vesicle trafficking performs essential functions in eukaryotic cells, such as membrane trafficking and delivery of molecules to their destinations. A major endocytotic route in plants is vesicle trafficking to the vacuole that plays an important role in plant salt tolerance. The final step in this pathway is mediated by the AtVAMP7C family of vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNAREs) that carry out the vesicle fusion with the tonoplast. Exposure to high-salt conditions causes immediate ionic and osmotic stresses, followed by production of reactive oxygen species. Here, we show that the reactive oxygen species are produced intracellularly, in endosomes that were targeted to the central vacuole. Suppression of the AtVAMP7C genes expression by antisense AtVAMP711 gene or in mutants of this family inhibited fusion of H2O2-containing vesicles with the tonoplast, which resulted in formation of H2O2-containing megavesicles that remained in the cytoplasm. The antisense and mutant plants exhibited improved vacuolar functions, such as maintenance of DeltapH, reduced release of calcium from the vacuole, and greatly improved plant salt tolerance. The antisense plants exhibited increased calcium-dependent protein kinase activity upon salt stress. Improved vacuolar ATPase activity during oxidative stress also was observed in a yeast system, in a DeltaVamp7 knockout strain. Interestingly, a microarray-based analysis of the AtVAMP7C genes showed a strong down-regulation of most genes in wild-type roots during salt stress, suggesting an evolutionary molecular adaptation of the vacuolar trafficking.

Arabidopsis↗

Crystal structure of a major fragment of the salt-tolerant glutaminase from Micrococcus luteus K-3.

Glutaminase of Micrococcus luteus K-3 (intact glutaminase; 48kDa) is digested to a C-terminally truncated fragment (glutaminase fragment; 42kDa) that shows higher salt tolerance than that of the intact glutaminase. The crystal structure of the glutaminase fragment was determined at 2.4A resolution using multiple-wavelength anomalous dispersion (MAD). The glutaminase fragment is composed of N-terminal and C-terminal domains, and a putative catalytic serine-lysine dyad (S64 and K67) is located in a cleft of the N-terminal domain. Mutations of the S64 or K67 residues abolished the enzyme activity. The N-terminal domain has abundant glutamic acid residues on its surface, which may explain its salt-tolerant mechanism. A diffraction analysis of the intact glutaminase crystals (a twinning fraction of 0.43) located the glutaminase fragment in the unit cell but failed to turn up clear densities for the missing C-terminal portion of the molecule.

Bacterial Proteins↗

Specificity for Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate of Nitrate Reductase from the Salt-tolerant Alga Dunaliella parva.

Nitrate reductase of the salt-tolerant alga Dunaliella parva could utilize NADPH as well as NADH as an electron donor. The two pyridine nucleotide-dependent activities could not be separated by either ion exchange chromatography on DEAE-cellulose or gel filtration on Sepharose 4B. The NADPH-dependent activity was not inhibited by phosphatase inhibitors. NADPH was not hydrolyzed to NADH and inorganic phosphate in the course of nitrate reduction. Reduction of nitrate in vitro could be coupled to a NADPH-regenerating system of glycerol and NADP-dependent glycerol dehydrogenase. It is concluded that the nitrate reductase of D. parva will function with NADPH as well as NADH. This is a unique characteristic not common to most algae.

Journal Article↗

Yeast putative transcription factors involved in salt tolerance.

Four putative yeast transcription factors (Hal6-9p) have been identified which upon overexpression in multicopy plasmids increase sodium and lithium tolerance. This effect is mediated, at least in part, by increased expression of the Enalp Na+/Li+ extrusion pump. Hal6p and Hal7p are bZIP proteins and their gene disruptions affected neither salt tolerance nor ENA1 expression. Hal8p and Hal9p are putative zinc fingers and their gene disruptions decreased both salt tolerance and ENA1 expression. Therefore, Hal8p and Hal9p, but not Hal6p and Hal7p, qualify as transcriptional activators of ENA1 under physiological conditions. Hal8p seems to mediate the calcineurin-dependent part of ENA1 expression.

Adaptation, Physiological↗

Adaptation to salt stress in a salt-tolerant strain of the yeast Yarrowia lipolytica.

We have studied the cellular mechanisms underlying adaptation to salt stress in a newly isolated osmo- and salt-tolerant strain of the yeast Yarrowia lipolytica. When cells are incubated in the presence of 9% NaCl, a rapid change in their size and shape is observed. Salt stress is accompanied by an increase in the intracellular level of glycerol, free amino acids (notably proline and aliphatic amino acids), and Na+, as well as by changes in lipid and fatty acid composition.

Atriplex↗

Direct interaction of a divergent CaM isoform and the transcription factor, MYB2, enhances salt tolerance in arabidopsis.

Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of enzymes and proteins. Plants express numerous CaM isoforms that exhibit differential activation and/or inhibition of CaM-dependent enzymes in vitro. However, the specific biological functions of plant CaM are not well known. In this study, we isolated a cDNA encoding a CaM binding transcription factor, MYB2, that regulates the expression of salt- and dehydration-responsive genes in Arabidopsis. This was achieved using a salt-inducible CaM isoform (GmCaM4) as a probe from a salt-treated Arabidopsis expression library. Using domain mapping, we identified a Ca2+-dependent CaM binding domain in MYB2. The specific binding of CaM to CaM binding domain was confirmed by site-directed mutagenesis, a gel mobility shift assay, split ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Interestingly, the specific CaM isoform GmCaM4 enhances the DNA binding activity of AtMYB2, whereas this was inhibited by a closely related CaM isoform (GmCaM1). Overexpression of Gm-CaM4 in Arabidopsis up-regulates the transcription rate of AtMYB2-regulated genes, including the proline-synthesizing enzyme P5CS1 (Delta1-pyrroline-5-carboxylate synthetase-1), which confers salt tolerance by facilitating proline accumulation. Therefore, we suggest that a specific CaM isoform mediates salt-induced Ca2+ signaling through the activation of an MYB transcriptional activator, thereby resulting in salt tolerance in plants.

Arabidopsis↗