In vitro salt tolerance of cell wall enzymes from halophytes and glycophytes

The halophyte Suaeda maritima grows optimally in high concentrations(40–60% seawater) of salt. In these conditions the concentrationof salt in the apoplast of the leaves is at least 500 mM, aconcentration which severely inhibits the activity of cytoplasmicenzymes of both glycophytes and halophytes. The in vitro salttolerance of a number of cell wall enzymes was assayed in thepresence of a range of concentrations of NaCl. There was nosignificant inhibition of the activity of galactosidase, glucosidase,peroxidase or xyloglucan endo-transglycosylase extracted fromSuaeda maritima by in vitro concentrations of NaCl up to atleast 1 M. In vitro salt tolerance of cell wall enzymes wasnot restricted to the halophyte, similar enzymes from the non-halophilicrelative Kochia tricophylla, and from the glycophytes Vignaradiata and Cicer arietinum, were inhibited little, or not atall, by the same concentrations of salt. Pectin esterase wassomewhat less tolerant, but activity at 500 mM NaCl was stillgreater than at 0 mM NaCl in both Suaeda and Vigna. It is concludedthat these enzymes of the cell wall compartment are much moresalt-tolerant than cytoplasmic enzymes of higher plants. Theresults are discussed in relation to conditions thought to pertainin the apoplast. Key words: Apoplast, cell wall enzymes, halophyte, salt tolerance, Suaeda maritima     

Lipolytic activity from Halobacteria: screening and hydrolase production

Strains of Halobacteria from an Algerian culture collection were screened for their lipolytic activity against p-nitrophenyl butyrate (PNPB) and p-nitrophenyl palmitate (PNPP). Most strains were active on both esters and 12% hydrolyzed olive oil. A strain identified as Natronococcus sp. was further studied. It grew optimally at 3.5 M NaCl, pH 8 and 40 degrees C. An increase in temperature shifted the optimum salt concentration range for growth from a wider range of 2-4 M, obtained at 25-30 degrees C, to a narrower range of 3.5-4 M, obtained at 35-40 degrees C. At 45 degrees C the optimum salt concentration was 2 M. These results show a clear correlation between salt and temperature requirement. The optimum conditions for the production of hydrolytic activity during growth were: 3.5 M NaCl and pH 8 for PNPB hydrolytic activity and 4 M NaCl and pH 7.5 for PNPP hydrolytic activity; both at 40 degrees C. The clear supernatant of cells grown at 4 M NaCl showed olive oil hydrolysis activity (in presence of 4 M NaCl) demonstrating the occurrence of a lipase activity in this strain. To our knowledge, this is the first report of a lipase activity at such high salt concentration.     

Cell wall modifications during osmotic stress in Lactobacillus casei

AIMS: To study the modification of the cell wall of Lactobacillus casei ATCC 393 grown in high salt conditions. METHODS AND RESULTS: Differences in the overall structure of cell wall between growth in high salt (MRS + 1 mol l(-1) NaCl; N condition) and control (MRS; C condition) conditions were determined by transmission electronic microscopy and analytical procedures. Lactobacillus casei cells grown in N condition were significantly larger than cells grown under unstressed C condition. Increased sensitivity to mutanolysin and antibiotics with target in the cell wall was observed in N condition. Purified cell wall also showed the increased sensitivity to lysis by mutanolysin. Analysis of peptidoglycan (PG) from stressed cells showed that modification was at the structural level in accordance with a decreased PG cross-link involving penicillin-binding proteins (PBP). Nine PBP were first described in this species and these proteins were expressed in low percentages or presented a modified pattern of saturation with penicillin G (Pen G) during growth in high salt. Three of the essential PBP were fully saturated in N condition at lower Pen G concentrations than in C condition, suggesting differences in functionality in vivo. CONCLUSIONS: The results show that growth in high salt modified the structural properties of the cell wall. SIGNIFICANCE AND IMPACT OF STUDY: Advances in understanding the adaptation to high osmolarity, in particular those involving sensitivity to lysis of lactic acid bacteria.     

Effect of high salinity on tracheary element differentiation in light-grown callus of Mesembryanthemum crystallinum

This study investigated the inhibitory effects of NaCl on tracheary element (TE) differentiation in light-grown callus of ice plant Mesembryanthemum crystallinum L., a halophyte which adaptes well to saline environments. When ice plant callus was grown in a modified Linsmaier-Bednar and Skoog culture medium containing no NaCl (control medium), up to 20% of ice plant cells differentiated into tracheary elements during in vitro culture. Close examination of callus tissues stained with potassium permanganate revealed that tracheary elements were aggregated as discrete nodules. Some strikingly elongated tracheary elements were found in the macerated tissues. Experimental results indicated that adding 200 mM NaCl to the control medium reversibly inhibited the formation of tracheary element in the halophytic cells. The rate of tracheary element formation increased accordingly as the rate of cell growth in control medium. In the presence of high salt, the degree of tracheary element differentation remained low through the growth cycle. The inhibitory effect of salt on tracheary element differentiation was overcome by adding 10 mg l⁻¹ salicylic acid, a known signaling compound that induces a diverse group of defense-related genes, including genes involved in reinforcing the host cell wall. Furthermore, microscopic examination revealed that most tracheary elements formed under this treatment (200 mM NaCl plus 10 mg l⁻¹ salicylic acid) were round shaped. The results suggest that high salt inhibits both the biosynthesis of secondary wall components and cell elongation ice plant in vitro culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

不同能源柳无性系对NaCl胁迫的生理响应

为了探索不同能源柳无性系的耐盐潜力,在盆栽条件下,对1年生能源柳无性系2、4、C、E扦插苗设置NaCl盐分梯度,使其土壤含盐量保持在0、0.2%、0.4%、0.6%、0.8%,并以乡土旱柳为对照,胁迫45d后,对5个无性系的气体交换参数、保护酶活性和有机渗透调节物质进行测定。研究发现:(1)在低浓度盐胁迫下(土壤含盐量0 0.4%),导致各能源柳光合速率降低的是气孔因素;在高浓度盐胁迫下(土壤含盐量大于0.4%),导致各能源柳无性系光合速率下降的主要是非气孔因素。(2)随着土壤盐浓度的增加,各能源柳无性系的过氧化氢酶(CAT)活性逐渐减小,过氧化物酶(POD)活性先增加后减小,而超氧化物歧化酶(SOD)活性呈现增加的趋势,并且4个能源柳无性系在盐胁迫条件下保护酶活性强于旱柳。(3)在土壤含盐量小于0.6%时,4个能源柳无性系的可溶性蛋白含量始终大于旱柳;在整个胁迫过程中,能源柳C的脯氨酸含量始终大于旱柳。(4)与旱柳相比,4个能源柳无性系在土壤含盐量0 0.4%的土壤中都能良好生长,其中能源柳C的耐盐潜力更大,可在土壤含盐量为0.6%的土壤中正常生长。这说明,4个能源柳无性系均可在天津轻、中度盐渍化地区栽培。     

Genotypic variation of nodules’ enzymatic activities in symbiotic nitrogen fixation among common bean (Phaseolus vulgaris L.) genotypes grown under salinity constraint

The effect of salt stress, under glasshouse conditions, was studied on plant biomass, nodulation, and activities of acid phosphatases (APase, EC 3.1.3.2) and trehalose 6-phosphate phosphatase (TPP, EC 3.1.3.12) in the symbiosis common bean (Phaseolus vulgaris L.)-rhizobia nodules. Four common bean recombinant inbred lines (147, 115, 104 and 83) were separately inoculated, with CIAT 899 or RhM11 strains and grown in hydroaeroponic culture. Two NaCl levels (0 and 25 mM NaCl plant^?1 week^?1 corresponding, respectively, to the control and the salt treatment) were applied and the culture was assessed during 42 days after their transplantation. The results showed that the nodulation of these lines was not affected by salinity except for the line 83 inoculated with CIAT 899, whose nodule dry weight decreased by 48.24 % compared with the corresponding controls. For the other symbiotic combinations, shoot and root biomasses were not significantly affected by salt constraint. Salinity stress generally reduced acid phosphatise and trehalose phosphate phosphatase activities in nodules that were less affected in plants inoculated with RhM11. Based on our data, it appears that nodule phosphatase activity may be involved in salinity tolerance in common beans and the levels of salt tolerance depend principally on specific combination of the rhizobial strain and the host cultivar.     

Effect of extreme salt concentrations on the physiology and biochemistry of Halobacteroides acetoethylicus.

Halobacteroides acetoethylicus grew in media with 6 to 20% NaCl and displayed optimal growth at 10% NaCl. When grown in medium with an [NaCl] of 1.7 M, the internal cytoplasmic [Na+] and [Cl-] were 0.92 and 1.2 M, respectively, while K+ and Mg2+ concentrations in cells were 0.24 and 0.02 M, respectively. Intracellular [Na+] was fourfold higher than intracellular [K+]. Since Na+ and Cl- ions were not excluded from the cell, the influence of high salt concentrations on key enzyme activities was investigated in crude cell extracts. Activities greater than 60% of the maximal activity of the following key catabolic enzymes occurred at the following [NaCl] ranges: glyceraldehyde-3-phosphate dehydrogenase, 1 to 2 M; alcohol dehydrogenase (NAD linked), 2 to 4 M; pyruvate dehydrogenase, 0.5 to 1 M; and hydrogenase (methyl viologen linked), 0.5 to 3 M. These studies support the hypothesis that obligately halophilic, anaerobic eubacteria adapt to extreme salt concentrations differently than do halophilic, aerobic eubacteria, because they do not produce osmoregulants or exclude Cl-. This study also demonstrated that these halophilic, anaerobic eubacteria have a physiological similarity to archaebacterial halophiles, since Na+ and Cl- are present in high concentrations and are required for enzymatic activity.     

Amino acid composition of bulk protein and salt relationships of selected enzymes of Salinibacter ruber,an extremely halophilic bacterium

The extremely halophilic bacterium Salinibacter ruber was previously shown to have a high intracellular potassium content, comparable to that of halophilic Archaea of the family Halobacteriaceae. The amino acid composition of its bulk protein showed a high content of acidic amino acids, a low abundance of basic amino acids, a low content of hydrophobic amino acids, and a high abundance of serine. We tested the level of four cytoplasmic enzymatic activities at different KCl and NaCl concentrations. Nicotinamide adenine dinucleotide (NAD)-dependent isocitrate dehydrogenase functioned optimally at 0.5-2 M KCl, with rates of 60% of the optimum value at 3.3 M. NaCl provided less activation: 70% of the optimum rates in KCl were found at 0.2-1.2 M NaCl, and above 3 M NaCl, activity was low. We also detected nicotinamide adenine dinucleotide phosphate (NADP)-dependent isocitrate activity, which remained approximately constant between 0-3.2 M NaCl and increased with increasing KCl concentration. NAD-dependent malate dehydrogenase functioned best in the absence of salt, but rates as high as 25% of the optimal values were measured in 3-3.5 M KCl or NaCl. NAD-dependent glutamate dehydrogenase, assayed by the reductive amination of 2-oxoglutarate, showed low activity in the absence of salt. NaCl was stimulatory with optimum activity at 3-3.5 M. However, no activity was found above 2.5 M KCl. Although the four activities examined all function at high salt concentrations, the behavior of individual enzymes toward salt varied considerably. The results presented show that Salinibacter enzymes are adapted to function in the presence of high salt concentrations.     

Development of NaCl-tolerant line in Chrysanthemum morifolium Ramat. through shoot organogenesis of selected callus line

Plants were regenerated successfully through shoot organogenesis of a NaCl-selected callus line of Chrysanthemum morifolium Ramat. cv. Maghi Yellow (a salt sensitive cultivar), developed through stepwise increase in NaCl concentration (0-100mM) in the MS medium. The stepwise increase in NaCl concentration from a relatively low level to cytotoxic level was found to be a better way to isolate NaCl-tolerant callus line, since direct transfer of callus to high saline medium was detrimental to callus survival and growth. The selected callus line exhibited significant increase in superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities compared to control callus (grown in medium devoid of NaCl). Stability of salt tolerance character of the selected callus line was checked by growing the calli in NaCl-free medium for 3 consecutive months followed by re-exposure to higher salinity stress (120mM NaCl). Among different growth regulator treatments, a combination of 5mgl(-1) TDZ (Thidiazuron) along with 0.25mgl(-1) NAA and 0.5mgl(-1) GA(3) was found to be the most effective for shoot organogenesis in selected callus line. The regeneration potential of the NaCl-tolerant callus ranged from 20.8% to 0% against 62.4% to 0% in control callus line. Under elevated stress condition (medium supplemented with 250mM NaCl), selected calli derived regenerants (S1 plants) exhibited significantly higher SOD and APX activities over both PC (positive control: control callus derived plants grown on MS medium devoid of NaCl) and NC (negative control: control callus derived plants subjected to 250mM NaCl stress) plants. In addition, the NC plants showed stunted growth, delayed root initiation, and had lesser number of roots as compared to S1 plants. Based on growth performance and antioxidant capacity, the S1 plants could be considered as NaCl-tolerant line showing all positive adaptive features towards the salinity stress. Further study on agronomic performance of these S1 plants under saline soil condition need to be undertaken to check the genetic stability of the induced salt-tolerance.     

Production of halostable β-mannanase and β-mannosidase by strain NN, a new extremely halotolerant bacterium

An extremely halotolerant mannan-degrading bacterium (strain NN) was isolated from the Great Salt Lake, Utah, USA. Strain NN grew at salinities from 0 to 20% NaCl with optimal growth at 0% NaCl. When grown on 0.2% (w/v) locust bean gum as the carbon source at 10% NaCl, both β-mannanase and β-mannosidase activities were produced. β-Mannosidase activity was shown to be cell-associated, while at least 23% of the total β-mannanase activity was extracellular. The optimum temperature and pH for β-mannanase activity were 70 °C and 7.6, and for β-mannosidase 25 °C and 7.0. The β-mannanase system retained full activity after 24 h of incubation at 60 °C and 10% NaCl. β-Mannanase activity was maximal at 1% NaCl and β-mannosidase activity at 0.5% NaCl. Despite these low salinity optima, 50% and 100% respectively of the initial β-mannanase and β-mannosidase activities remained after 48 h of incubation at 20% NaCl, indicating a high degree of halostability. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis revealed the presence of at least eight different mannan-degrading proteins in the cell-free culture supernatant of cultures grown on locust bean gum. Received: 19 March 1998 /  Received revision: 8 June 1998 / Accepted: 14 June 1998     

Extracellular cell wall lytic enzyme from Staphylococcus aureus: purification and partial characterization

An autolysin obtained from culture fluid of Staphylococcus aureus strain 8507 was purified 3,000-fold. One milligram of this preparation (S-5DL) will solubilize 12 mg of cell wall in 1 hr. The major activity is N-acetylmuramyl-l-alanine amidase. Recovery of lytic activity in the purified preparation was repeatably only 20% of the starting level. This suggests that other cell wall lytic enzymes may be present in the starting material. The S-5DL enzyme has been compared to freeze-thaw extracted enzyme (AFZ). Both enzymes precipitate in 0.01 m KPO(4) (pH 6.0) and dissolve in 0.1 to 0.7 m NaCl. Fifty per cent of the AFZ activity and 66% of the S-5DL activity bind rapidly to cell walls of S. aureus at 0 C in the presence of magnesium ion. None of the AFZ activity and 66% of the S-5DL activity bind to cell walls at 0 C in the absence of magnesium ion. The cell walls of nine different strains of S. aureus were compared for level of native autolysin activity. These same walls after inactivation of the native autolysin were tested for susceptibility to the S-5DL enzyme.     

Immunization of Mice with Components of Pasteurella multocida

Log-phase cells of Pasteurella multocida strain P-1059 were used to prepare isolated culture filtrate, cell wall, and cytoplasmic components. Culture filtrate was further separated by column chromatography. A portion of cytoplasm and culture filtrate was conjugated to ferritin by means of metaxylylene diisocyanate. Cell walls induced more protection in mice than the conjugated or unconjugated cytoplasm or culture filtrate. The cell walls caused edema and erythema when given intradermally in rabbits, whereas cytoplasm and culture filtrate produced dermal necrosis. The first of four chromatographically separated fractions of culture filtrate was possibly more immunogenic in mice than cell walls. This fraction was less reactive intradermally in rabbits than cell walls but more reactive than the other fractions.     

Effects of NaCl and Proline on Polyphenol Oxidase Activity in Bean Seedlings

In this work, the effects of NaCl (0, 50, 100, and 150 mM), proline (0, 5 and 10 mM) and NaCl + proline in combinations on activity of polyphenol oxidase (PPO; E.C. 1.10.3.1) and soluble protein content have been investigated in the root, stem and leaf tissues of bean (Phaseolus vulgaris L.) seedlings grown in embryo culture. PPO activities were higher in all the tissues treated with NaCl, proline and NaCl + proline combinations those that of the control tissues. The protein content was very high in tissues exposed to proline and NaCl + proline combination, but NaCl alone decreased protein contents in root and leaf tissues. The results suggest that proline may play a role as an enzyme-stabilizing agent in salt stress.     

Salt Tolerance of In Vitro Established Salt-Tolerant Rice Plants during Further Growth in Soil

In vitro salt tolerant rice plants established by step up treatment with 0.5, 1.0, 1.5 and 2.0 % NaCl at 3-week intervals were examined to determine whether they could grow in potted paddy soil containing 0, 0.55 or 0.75 % NaCl till harvesting. All the control plants were necrotic by the 4^th week in the culture. At the 10^th week of culture, 100 % of the salt-tolerant plants subjected to 0 or 0.55 % NaCl survived, and 78 % of the plants at 0.75 % NaCl. The Na⁺ and Cl^– contents in the leaves of salt-tolerant plants grown at 0.55 and 0.75 % NaCl were about 4 times of those without NaCl. The ion contents in non-tolerant plants and seedling plants were 10 to 12 times of those in 0 % NaCl treatment. One of the hypotheses to explain the present data is that the in vitro step up salt selection induces the capability to maintain no lethal concentration of NaCl in the leaves.     

A Biolchemical and Cytochemical Study of Peroxidase Activity in Roots of Pisum sativum: I. A COMPARISON OF DAB-PEROXIDASE AND GUAIACOL-PEROXIDASE WITH PARTICULAR EMPHASIS ON THE PROPERTIES OF CELL WALL ACTIVITY

Peroxidase activity in roots of Pisum sativum has been examinedusing both guaiacol and 3,3'-diaminobenzidine (DAB) as hydrogendonors. Biochemically, differences were observed between thetwo donors with respect to the pH optimum (6–9 and 4–0,respectively), and in response to added NaCl (guaiacol-peroxidasewas unaffected while the DAB-peroxidase was markedly inhibited).Both reactions showed highest specific activity in a high speedsupernatant fraction, and, of nine anionic bands demonstratedby gel electrophoresis with DAB, only six were visible withguaiacol. Histochemically, similar staining patterns were observedwith both donors. Cell wall fractions prepared by bead filtration contained 2%and 3.5% of the total peroxidase and acid phosphatase activitiesrespectively. 50% and 27% of these activities were ionicallybound, as indicated by salt treatment In addition, washing withsalt solutions produced a marked stimulation of peroxidase activityat high salt concentrations: this affect was not observed withthe supernatant peroxidase or with cell wall acid phosphatase.Possible functions of cell wall peroxidase are discussed     

Isolation of an osmotolerant ale strain of Saccharomyces cerevisiae

Saccharomyces cerevisiae (ale strain) grown in batch culture to stationary phase was tested for its tolerance to heat (50 degrees C for 5 min), hydrogen peroxide (0.3 M) and salt (growth in 1.5 M sodium chloride/YPD medium). Yeast cells which have been exposed previously to heat shock are more tolerant to hydrogen peroxide and high salt concentrations (1.5 M NaCl) than the controls. Their fermentative activity as judged by glucose consumption and their viability, as judged by cell number and density have higher levels when compared with cells not previously exposed to heat shock. Experimental conditions facilitated the isolation of S. cerevisiae ale strain, which was tolerant to heat, and other agents such as hydrogen peroxide and sodium chloride.     

Different antioxidant responses to salt stress in two different provenances of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> L.

Seedlings of two Tunisian Carthamus tinctorius L. provenances (Kairouan and Tazarka) differing in salt sensitivity were hydroponically grown at 0 and 50 mM NaCl over 21 days. Leaves of Kairouan (salt-sensitive) showed a 48% restriction in their growth at 50 mM NaCl although they accumulated less sodium than those of Tazarka (less salt-sensitive) that maintained an unchanged growth. Salt treatment induced oxidative stress in C. tinctorius and the effect was more pronounced in the leaves of the more salt sensitive provenance, Kairouan. Both provenances exhibited a stimulation of antioxidant enzyme activities with higher catalase (CAT) and superoxide dismutase (SOD) activities in Tazarka and higher peroxidase (POD) activity in Kairouan. But, it seems that antioxidant activities were more correlated with polyphenol content. Actually, leaves of Tazarka experienced higher polyphenol and antioxidant activity than Kairouan at 50 mM NaCl. Hence, moderate salinity (3 g NaCl L⁻¹) enhanced bioactive molecule yield in the less salt sensitive provenance, Tazarka. In addition, C. tinctorius was found rich in ascorbic acid, but the moderate salt stress enhanced its production only in the sensitive provenance.     

Characterization of Lignocellulolytic Activities from a Moderate Halophile Strain of Aspergillus caesiellus Isolated from a Sugarcane Bagasse Fermentation

A moderate halophile and thermotolerant fungal strain was isolated from a sugarcane bagasse fermentation in the presence of 2 M NaCl that was set in the laboratory. This strain was identified by polyphasic criteria as Aspergillus caesiellus. The fungus showed an optimal growth rate in media containing 1 M NaCl at 28°C and could grow in media added with up to 2 M NaCl. This strain was able to grow at 37 and 42°C, with or without NaCl. A. caesiellus H1 produced cellulases, xylanases, manganese peroxidase (MnP) and esterases. No laccase activity was detected in the conditions we tested. The cellulase activity was thermostable, halostable, and no differential expression of cellulases was observed in media with different salt concentrations. However, differential band patterns for cellulase and xylanase activities were detected in zymograms when the fungus was grown in different lignocellulosic substrates such as wheat straw, maize stover, agave fibres, sugarcane bagasse and sawdust. Optimal temperature and pH were similar to other cellulases previously described. These results support the potential of this fungus to degrade lignocellulosic materials and its possible use in biotechnological applications.