NaCl effects on proline metabolism in rice (Oryza sativa) seedlings

Salt-stress effects on osmotic adjustment, ion and proline concentrations as well as proline metabolizing enzyme activities were studied in two rice ( Oryza sativa L.) cultivars differing in salinity resistance: I Kong Pao (IKP; salt-sensitive) and Nona Bokra (salt-resistant). The salt-sensitive cultivar exposed to 50 and 100 m M NaCl in nutritive solution for 3 and 10 days accumulated higher levels of sodium and proline than the salt-resistant cultivar and displayed lower levels of osmotic adjustment. Proline accumulation was not related to proteolysis and could not be explained by stress-induced modifications in Δ¹-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) or proline dehydrogenase (PDH; EC 1.5.1.2) activities recorded in vitro. The extracted ornithine Δ -aminotransferase (OAT; EC 2.6.1.13) activity was increased by salt stress in the salt-sensitive cultivar only. In both genotypes, salt stress induced an increase in the aminating activity of root glutamate dehydrogenase (GDH; EC 1.4.1.2) while deaminating activity was reduced in the leaves of the salt-sensitive cultivar. The total extracted glutamine synthetase activity (GS; EC 6.3.1.2) was reduced in response to salinity but NaCl had contrasting effects on GS1 and GS2 isoforms in salt-sensitive IKP. Salinity increased the activity of ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) extracted from leaves of both genotypes and increased the activity of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in the salt-sensitive cultivar. It is suggested that proline accumulation is a symptom of salt-stress injury in rice and that its accumulation in salt-sensitive plants results from an increase in OAT activity and an increase in the endogenous pool of its precursor glutamate. The physiological significance of the recorded changes are analyzed in relation to the functions of these enzymes in plant metabolism.     

Isolation and characterization of salt-sensitive mutants of a salt-tolerant yeast Zygosaccharomyces rouxii

Abstract Twenty salt-sensitive (ss) mutants were isolated from the salt-tolerant yeast Zygosaccharomyces rouxii by treatment with N -methyl- N '-nitro- N -nitrosoguanidine. The mutants were divided into five classes on the basis of their ability to grow in media containing various high concentrations of NaCl. The mutant with the greatest sensitivity to NaCl of all the mutants tested was able to grow very slowly with a longer lag phase in medium containing 2 M NaCl, in contrast to the wild strain which had the capacity to grow in medium containing 3.5 M NaCl. Most of the ss mutants exhibited, to some extent, less tolerance to high concentrations of glucose than the wild strain. It appeared from the characterization of the ss mutants that the following factors are necessary for growth of Z. rouxii in high concentrations of NaCl: (a) the ability to produce glycerol under these conditions; (b) the ability to maintain a defined concentration of glycerol within the cells; (c) the ability to take up glycerol that has leaked into the medium, and to assimilate glycerol; and (d) unknown factor(s).     

Contribution of chemical changes in membrane lipids to the osmoadaptation of the halophilic bacterium Chromohalobacter salexigens

The long-term response of the broad-salt growing halophile Chromohalobacter salexigens DSM 3043T to salt stress has been investigated with respect to adaptive changes in membrane lipid composition. This study included the wild-type and three salt-sensitive, ectoine-deficient strains: CHR62 (ectA::Tn1732, unable to grow above 0.75 M NaCl), CHR63 (ectC::Tn1732, unable to grow above 1.5 M NaCl), and CHR64, which was able to grow in minimal medium M63 up to 2.5 M NaCl, but its growth was slower than the wild-type strain at salinities above 1.5 M NaCl. This mutant accumulated ectoine and hydroxyectoine as major compatible solutes, but also the ectoine precursor, N-gamma-acetyldiaminobutyric acid, and was found to be affected in the ectoine synthase gene ectC. The main phospholipids of the wild-type strain were phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin (CL). Major fatty acids were detected as 16:0, 18:1, and 16:1, including significant amounts of cyc-19:0, and cyc-17:0. CL and cyclopropane fatty acids (CFA) levels were elevated when the wild-type strain was grown at high salinity (2.5 M NaCl). Membranes of the most salt-sensitive trains CHR62 and CHR63, but not of the less salt-sensitive strain CHR64, contained lower levels of CL. The proportion of cyc-19:0 in CHR64 was three-fold (at 2.0M NaCl) and 2.5-fold (at 2.5 M NaCl) lower than that of the wild type, suggesting that this mutant has a limited capacity to incorporate CFA into phospholipids at high salt. The addition of 1 mM ectoine to cultures of the wild-type strain increased the ratio PG/CL from 1.8 to 3.3 at 0.75 M NaCl, and from 1 to 6.5 at 2.5 M NaCl, and led to a slight decrease in CFA content. Addition of 1 mM ectoine to the mutants restored the steady-state levels of CL and CFA found in the wild-type strain supplemented with ectoine. These findings suggest that exogenous ectoine might attenuate the osmostress response involving changes in membrane lipids.     

Stress responses of tobacco cells to high temperature and salinity. Proline accumulation and phosphorylation of polypeptides

Stress responses to high temperatures (35–48°C) and salinity (170–340 mM NaCl) and thermotolerance were investigated for the salt-sensitive wild type and the salt-tolerant N_rE_s-1 strain of Nicotiana sylvestris L. using suspension-cultured cells. Under saline conditions, N_rE_s-1 strain cells accumulated proline, polyamine (putrescine and spermidine) and betaine in contrast to wild-type cells. The simultaneous treatment of salt-tolerant cells with high temperature (40°C) and NaCl (170 or 340 mM ) resulted in a transient overproduction of proline accompanied by an increase in their thermotolerance. At the high temperature, the synthesis of polypeptides and the accumulation of heat shock protein HSP70 mRNA were not affected by salinity. The higher thermotolerance of the NaCl-tolerant cells could not be related to osmoprotecting sugar-starch interconversions but could rather be associated with selective phosphorylation of several polypeptides (23–24, 27, 31–32, 47 kDa) prior to the accumulation of proline. The possible role of polyamines and polypeptide phosphorylation in this respect is discussed.     

NaCl和等渗聚乙二醇对苹果属植物游离脯氨酸含量的影响

测定了不同耐盐性苹果属植物珠眉海棠、小金海棠和山定子幼苗各部位的游离脯氨酸含量,结果表明,NaCl胁迫下苹果属植物游离脯氨酸含量增加均大于等渗PEG处理,NaCl和等渗PEG处理对耐盐种的游离脯氨酸含量影响较小,高盐度下盐敏感种的游离脯氨酸含量持续大量增加。     

Adaptational changes in Staphylococcus aureus MF31 grown above its maximum temperature when protected by NaCl: physiological studies

Staphylococcus aureus MF31 can grow at 46 degrees C, 2 degrees C above its normal maximum temperature of growth if 1 M NaCl is added to the medium. In the present work we show that monosodium glutamate, proline, threonine, aspartic acid, and betaine (in order of decreasing effectiveness) also enabled cells to grow at 46 degrees C. Cells grown at 46 degrees C in he presence of salt (protected or P cells) accumulated glutamate more rapidly than cells grown at 37 degrees C without salt (normal or N cells) and contained an increased amino acid pool. The principal constituents of this pool were dicarboxylic amino acids and proline. Turbidimetric evidence suggests that NaCl caused plasmolysis in S. aureus. The P cells, although grown in 1 M NaCl, had about the same Cl- and K+ content as the N cells grown without added NaCl. P cells had increased heat resistance but high concentrations of CaCl2 in the heating menstruum reduced their D55 value from a maximum of 214 min to less than 30 s. We suggest that growth at 46 degrees C in 1 M NaCl can be explained, in part at least, by the increased amino acid pool internal to the cell and the external osmotic support given by Cl- anions excluded by the cell.     

Regulation of cytoplasmic proline levels in Salmonella typhimurium: effect of osmotic stress on synthesis, degradation, and cellular retention of proline.

I investigated the effects of osmotic stress on the synthesis and catabolism of proline in Salmonella typhimurium by measuring the intracellular and extracellular proline levels in various strains. In the wild-type strain, exposure to 0.8 M NaCl did not cause a significant change in the intracellular proline level; however, it brought about a 6.5-fold increase in the intracellular glutamate pool size. These results indicate that gamma-glutamyl kinase is inhibited by proline in wild-type cells in media of normal or elevated osmolarity. I also tested whether proline is subject to turnover in cells wild type with respect to the enzymes of the proline degradation pathway. In strains that were wild type for proline biosynthesis, the loss of the proline catabolic enzymes, due to putA mutations, did not result in a statistically significant increase in the intracellular proline levels. Therefore, in the wild-type strain, proline turnover does not seem to be important for control of the intracellular proline levels. However, in a proline-overproducing mutant, a putA lesion caused a threefold increase in the intracellular proline level and a 6.5-fold increase in the extracellular proline level, indicating that proline is subject to turnover in the overproducing mutant. The proline-overproducing mutants excreted large quantities of the proline into the culture medium; osmotic stress altered the partitioning of proline such that the ratio of intracellular to extracellular levels of proline increased with increased osmotic stress. The increased cellular retention of proline in media of high osmolarity is probably due to the functioning of the ProP and ProU proline transport systems, which are stimulated under conditions of osmotic stress.     

Mutational Engineering of the cyanobacterium Nostoc muscorum for resistance to growth-inhibitory action of LiCl and NaCl

The effect of NaCl on two vital processes of cyanobacterial metabolism, viz. N(2) fixation and oxygenic photosynthesis, was studied in the cyanobacterium Nostoc muscorum grown diazotrophically. An increase in NaCl concentration suppressed the formation of heterocyst and adversely affected the nitrogenase activity in the parent, whereas in Li(+)-R and Na(+)-R mutants NaCl stress did not cause any adverse effect. The rate of photosynthetic O(2)-evolution was also adversely affected by the NaCl stress, but the magnitude was less than that of nitrogenase activity. L-Proline, the well-known osmoprotectant, provided protection to the cyanobacterium against NaCl stress. The parent strain utilized L-proline as a nitrogen source and suppressed heterocyst formation and nitrogenase activity, while mutants showed normal heterocyst frequency and nitrogenase activity. Therefore, it may be that the proline metabolism is altered as a result of mutation. The intracellular levels of proline in the parent were enhanced about threefold in the medium containing 1 mol x m(-3) proline, while in mutants there was no significant increase in the intracellular level of proline. In the medium containing both NaCl and proline, the intracellular level of proline was enhanced in the parent as well as in both mutant strains. This suggests that the parent strain possessed both normal proline uptake and salt-induced proline uptake systems, whereas the mutant strains were defective in normal proline uptake and had only salt-induced proline uptake. The over-accumulation of proline in the presence of NaCl stress is due either to the loss of proline oxidase activity or to the accumulation of exogenous proline.     

Physiological characterization and stress-induced metabolic responses of Dunaliella salina isolated from salt pan

A Dunaliella strain was isolated from salt crystals obtained from experimental salt farm of the institute (latitude 21.46 N, longitude 72.11 degrees E). The comparative homology study of amplified molecular signature 18S rRNA, proves the isolated strain as D. salina. The growth pattern and metabolic responses such as proline, glycine betaine, glycerol, total protein and total sugar content to different salinity (from 0.5 to 5.5 M NaCl) were studied. The optimum growth was observed at 1.0 M NaCl and thereafter it started to decline. Maximum growth was obtained on 17th day of inoculation in all salt concentrations except 0.5 M NaCl, whereas maximum growth was observed on 13th day. There were no significant differences (P < 0.01) in chlorophyll a/b contents (1.0-1.16 +/- 0.05 mug chl. a and 0.2-0.29 +/- 0.01 mug chl. b per 10(6) cells) up to 2.0 M NaCl, however at 3.0 M NaCl a significant increase (2.5 +/- 0.12 mug chl. a and 0.84 +/- 0.4 mug chl. b per 10(6) cells) was observed which declined again at 5.5 M NaCl concentration (2.0 +/- 0.1 mug chl. a and 0.52 +/- 0.03 mug chl. b per 10(6) cells). Stress metabolites such as proline, glycine betaine, glycerol and total sugar content increased concomitantly with salt concentration. Maximum increase in proline (1.4 +/- 0.07 mug), glycine betaine (5.7 +/- 0.28 mug), glycerol (3.7 +/- 0.18 ml) and total sugar (250 +/- 12.5 mug) per 10(5) cells was observed in 5.5 M NaCl. A decrease in total protein with reference to 0.5 M NaCl was observed up to 3.0 M NaCl, however, a significant increase (P < 0.01) was observed at 5.5 M NaCl (0.19 +/- 0.01 mug per 10(5) cells). Inductive coupled plasma (ICP) analysis shows that intracellular Na(+) remained unchanged up to 2.0 M NaCl concentration and thereafter a significant increase was observed. No relevant increase in the intracellular level of K(+) and Mg(++) was observed with increasing salt concentration. Evaluation of physiological and metabolic attributes of Dunaliella salina can be used to explore its biotechnological and industrial potential.     

Effect of glutamate transport and catabolism on symbiotic effectiveness in Rhizobium leguminosarum bv. phaseoli

Seven Tn5 induced mutants unable to use glutamate as sole carbon and nitrogen source were isolated from the effective Rhizobium leguminosarum bv. phaseoli strain P121-R. As indicated by restriction and hybridisation analysis, all the mutants arose from a single Tn5 insertion in the chromosome. The ¹⁴C-glutamate uptake rate of the mutants was 76 to 88% lower than that of strain P121-R. Inoculation of Phaseolus vulgaris cv. Labrador with these mutants significantly decreased shoot dry matter yield and the total nitrogen content respectively, as compared to inoculation with the parental strain P121-R. All the mutants formed nodules, however they were smaller, white to greenish and approximately 30% less numerous than those formed by strain P121-R. These observations suggest that glutamate transport and catabolism in R. leguminosarum bv. phaseoli P121-R may play an important role in the establishment of an effective symbiosis in field bean. None of the mutants isolated was an auxotroph. All mutants were unable to grow on aspartate suggesting that glutamate and aspartate, probably have the same transporter as indicated in Rhizobium meliloti and in Bacillus subtilis. All mutants readily used glutamine, proline, arginine as sole carbon and nitrogen source, but grew more slowly than the wild type strain. On the other hand, all the mutants were impaired in growth on histidine and -aminobutyrate as sole carbon and nitrogen source. As the catabolism of these amino acids occurs predominantly through glutamate, our results indicate that mutants are also impaired in their ability to use histidine and -aminobutyrate as a nitrogen source. Our results also suggest that other amino acids catabolized through the glutamate pathways may be an additional important carbon source for bacteroids in nodules.     

不同耐盐性水稻幼苗根氨同化酶对盐胁迫的反应

在盐胁迫下,检测了耐盐性不同的水稻(Oryza sativa L.)品种根部氨同化酶及其相关参数的变化。结果表明,根的可溶性蛋白、谷氨酰胺合成酶(GS)及依赖于NADH的谷氨酸合酶(NADH-GOGAT)活性在高盐浓度下不同程度地降低,其影响大小依次为早花二号(盐敏感品种)、金珠一号(正常栽培品种)、津稻779(耐盐品种),与其耐盐性相一致。在盐胁迫条件下,在耐盐性较高的水稻品种中, GS和GOGAT活性比盐敏感品种高,NH4 浓度维持在较低的水平。Native-PAGE和活性染色结果表明,GSrb更容易受到外界环境的影响。在高浓度盐的胁迫下,早花二号、金珠一号的依赖于NADH的谷氨酸脱氢酶(NADH-GDH)活性都有较显著的升高,津稻779却无明显的变化,这和NH4 含量的变化相一致。盐不同程度地导致可溶性糖(TSS)在金珠一号和津稻779根部积累,而在早花2号的根部,可溶性糖的水平则随盐浓度的不同而表现出不同的变化。在所检测的品种中,脯氨酸的含量均有不同程度的升高,但在高盐浓度下,盐敏感品种的含量较低。这些结果提示,不同的水稻品种对盐胁迫的敏感程度与该品种GS以及GOGAT活性的高低有关。     

不同耐盐性水稻幼苗根氨同化酶对盐胁迫的反应

在盐胁迫下,检测了耐盐性不同的水稻(Oryza sativa L.)品种根部氨同化酶及其相关参数的变化.结果表明,根的可溶性蛋白、谷氨酰胺合成酶(GS)及依赖于NADH的谷氨酸合酶(NADH-GOGAT)活性在高盐浓度下不同程度地降低,其影响大小依次为早花二号(盐敏感品种)、金珠一号(正常栽培品种)、津稻779(耐盐品种),与其耐盐性相一致.在盐胁迫条件下,在耐盐性较高的水稻品种中,GS和GOGAT活性比盐敏感品种高,NH4 浓度维持在较低的水平.Native-PAGE和活性染色结果表明,GSrb更容易受到外界环境的影响.在高浓度盐的胁迫下,早花二号、金珠一号的依赖于NADH的谷氨酸脱氢酶(AADH-GDH)活性都有较显著的升高,津稻779却无明显的变化,这和NH4 含量的变化相一致.盐不同程度地导致可溶性糖(TSS)在金珠一号和津稻779根部积累,而在早花2号的根部,可溶性糖的水平则随盐浓度的不同而表现出不同的变化.在所检测的品种中,脯氨酸的含量均有不同程度的升高,但在高盐浓度下,盐敏感品种的含量较低.这些结果提示,不同的水稻品种对盐胁迫的敏感程度与该品种GS以及GOGAT活性的高低有关.     

Accumulation and role of compatible solutes in fast-growing Salinivibrio costicola subsp. yaniae

The moderately halophilic bacterium Salinivibrio costicola subsp. yaniae showed an extremely fast growth rate. Optimal growth was observed in artificial seawater containing 1.4 mol/L NaCl and in MM63 media containing 0.6 mol/L NaCl. We analyzed a variety of compatible solutes that had accumulated in this strain grown in the media. The supplementation effect of the compatible solutes glycine betaine, glutamate, and ectoine to the growth of S. costicola subsp. yaniae was examined. Glycine betaine and glutamate had no supplementation effect on the fast growth rate. Growth of salt-sensitive mutants MU1 and MU2, both of which were defective in the ability to synthesize ectoine, was not observed in MM63 medium in the presence of more than 1.0 mol/L NaCl. From these data, we conclude that ectoine was the predominant compatible solute synthesized in this bacterium that effected an extremely fast growth rate.     

Glutamate overcomes the salt inhibition of DNA polymerase III holoenzyme

Even though Escherichia coli can grow in media containing up to 1 M NaCl, one-fifth that amount of NaCl will completely inhibit the in vitro activity of DNA polymerase III holoenzyme. It has been established that the major intracellular ionic osmolytes are potassium and glutamate (Richey, B., Cayley, D. S., Mossing, M. C., Kolka, C., Anderson, C. F., Farrar, T. C., and Record, M. T., Jr. (1987) J. Biol. Chem. 262, 7157-7164). We have found that holoenzyme catalyzes replication efficiently in vitro in up to 1 M potassium glutamate. Two salt effects on the replication of single-stranded DNA were observed. At low salt replicative activity was enhanced and at high salt there was anion-specific inhibition. We have found that DNA polymerase III holoenzyme tolerated 10-fold higher concentrations of glutamate than chloride. The ability of various anions to extend the useful range of salt concentrations followed the order: phosphate less than chloride less than N-Ac-glutamate less than acetate less than glycine less than aspartate less than glutamate. With the exception of phosphate, this order followed the Hofmeister series indicating that the anion-specific effects were due to anions interacting at the protein-water interface at weak anion binding sites. Glutamate did not reverse the inhibition by chloride. The low salt enhancement and high salt inhibition effects were additive for the two anions indicating that they competed for common anion binding sites. The major salt-sensitive step was holoenzyme binding to template rather than the subsequent elongation reaction.     

Mutational Engineering of the Cyanobacterium <Emphasis Type="Italic">Nostoc muscorum</Emphasis> for Resistance to Growth-Inhibitory Action of LiCl and NaCl

The effect of NaCl on two vital processes of cyanobacterial metabolism, viz. N₂ fixation and oxygenic photosynthesis, was studied in the cyanobacterium Nostoc muscorum grown diazotrophically. An increase in NaCl concentration suppressed the formation of heterocyst and adversely affected the nitrogenase activity in the parent, whereas in Li⁺-R and Na⁺-R mutants NaCl stress did not cause any adverse effect. The rate of photosynthetic O₂-evolution was also adversely affected by the NaCl stress, but the magnitude was less than that of nitrogenase activity. L-Proline, the well-known osmoprotectant, provided protection to the cyanobacterium against NaCl stress. The parent strain utilized L-proline as a nitrogen source and suppressed heterocyst formation and nitrogenase activity, while mutants showed normal heterocyst frequency and nitrogenase activity. Therefore, it may be that the proline metabolism is altered as a result of mutation. The intracellular levels of proline in the parent were enhanced about threefold in the medium containing 1 mol m⁻³ proline, while in mutants there was no significant increase in the intracellular level of proline. In the medium containing both NaCl and proline, the intracellular level of proline was enhanced in the parent as well as in both mutant strains. This suggests that the parent strain possessed both normal proline uptake and salt-induced proline uptake systems, whereas the mutant strains were defective in normal proline uptake and had only salt-induced proline uptake. The over-accumulation of proline in the presence of NaCl stress is due either to the loss of proline oxidase activity or to the accumulation of exogenous proline. Received: 10 July 2002 / Accepted: 13 August 2002     

Use of the Mud(Ap,lac) bacteriophage to study the regulation of L-proline biosynthetic genes inEscherichia coli K-12

One operon fusion to the promoter of either theproA orproB genes of the proline biosynthetic pathway was obtained by the use of the Mud(Ap,lac) bacteriophage. This operon fusion was further stabilized by transformation with the plasmid pGW600 containing the wild type Mu repressor gene. The level of β-galactosidase in this strain was not affected by the presence of high concentrations of NaCl in the growth medium. Mutations affecting the regulation of thispro-lac genetic fusion were generated by the insertion of Tn5; β-galactosidase levels in these mutants were higher than in the parental strain when proline was present at a high level. In some of these mutants we observed either repression or maintenance of β-galactosidase levels whenpro-lac (F′proAB ⁺) merodiploids were constructed.     

Comparison of hup trait and intrinsic antibiotic resistance for assessing rhizobial competitiveness axenically and in soil

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.