Cold shock and cold acclimation proteins in the psychrotrophic bacterium Arthrobacter globiformis SI55.

The psychrotrophic bacterium Arthrobacter globiformis SI55 was grown at 4 and 25 degrees C, and the cell protein contents were analyzed by two-dimensional electrophoresis. Cells subjected to cold shocks of increasing magnitude were also analyzed. Correspondence analysis of protein appearance distinguished four groups of physiological significance. Group I contained cold shock proteins (Csps) overexpressed only after a large temperature downshift. Group II contained Csps with optimal expression after mild shocks. Group III contained proteins overexpressed after all cold shocks. These last proteins were also overexpressed in cells growing at 4 degrees C and were considered to be early cold acclimation proteins (Caps). Group IV contained proteins which were present at high concentrations only in 4 degrees C steady-state cells and appeared to be late Caps. A portion of a gene very similar to the Escherichia coli cspA gene (encoding protein CS7.4) was identified. A synthetic peptide was used to produce an antibody which detected a CS7.4-like protein (A9) by immunoblotting two-dimensional electrophoresis gels of A. globiformis SI55 total proteins. Unlike mesophilic microorganisms, this CS7.4-like protein was still produced during prolonged growth at low temperature, and it might have a particular adaptive function needed for balanced growth under harsh conditions. However, A9 was induced at high temperature by chloramphenicol, suggesting that CS7.4-like proteins have a more general role than their sole implication in cold acclimation processes.     

Differential accumulation of S-adenosylmethionine synthetase transcripts in response to salt stress

NaCl stress causes the accumulation of several mRNAs in tomato seedlings. An upregulated cDNA clone, SAM1, was found to encode a S-adenosyl-L-methionine synthetase enzyme (AdoMet synthetase). Expression of the cDNA SAM1 in a yeast mutant lacking functional SAM genes resulted in high AdoMet synthetase activity and AdoMet accumulation. We show that tomato plants contain at least four SAM isogenes. Clones corresponding to isogenes SAM2 and SAM3 have also been isolated and sequenced. they encode predicted polypeptides 95% and 92% identical, respectively, to the SAM1-encoded AdoMet Synthetase. RNA hybridization analysis showed a differential response of SAM genes to salt and other stress treatments. SAM1 and SAM3 mRNAs accumulated in the root in response to NaCl, mannitol or ABA treatments. SAM1 mRNA accumulated also in leaf tissue. These increases of mRNA level were apparent as soon as 8 h after the initiation of the salt treatment and were maintained for at least 3 days. A possible role for AdoMet synthetases in the adaptation to salt stress is discussed.     

Preferential accumulation of d-pinitol in Acrostichum aureum gametophytes in response to salt stress

Growth and metabolic responses to salt stress were studied in gametophytes of Acrostichum aureum L. (a mangrove fern). Gametophytes were cultured in 0–170 m M NaCl solutions. The growth of gametophytes was best in low NaCl concentrations (35 and 85 m M ), and was retarded at higher NaCl concentrations. Photosynthetic rate of gametophytes was also significantly reduced when grown in high NaCl concentrations. Severe salt stress (>120 m M NaCl) led to a preferential accumulation of d -pinitol in gametophytes, whereas the sporophyte accumulated d -1- O -methyl- muco -inositol. The content of d -pinitol reached up to 50% of the soluble carbohydrate pool of gametophytes under 155 and 170 m M NaCl. The accumulation of d -pinitol and other cyclitols in gametophytes was correlated with the retention of photochemical efficiency of photosystem II and the survival of gametophytes after transfer to solutions containing 340 and 600 m M NaCl, respectively.     

Sugar accumulation,photosynthesis and growth of two indica rice varieties in response to salt stress

Sugar, a final product of photosynthesis, is reported to be involved in the defense mechanisms of plants against abiotic stresses such as salinity, water deficiency, extreme temperature and mineral toxicity. Elements involved in photosynthesis, sugar content, water oxidation, net photosynthetic rate, activity of enzyme and gene expression have therefore been studied in Homjan (HJ), salt-tolerant, and Pathumthani 1 (PT1), salt-sensitive, varieties of rice. Fructose-1,6-biphosphatase (FBP) and fructokinase (FK) genes were rapidly expressed in HJ rice when exposed to salt stress for 1–6 h and to a greater degree than in PT1 rice. An increase in FBP enzyme activity was found in both roots and leaves of the salt-tolerant variety after exposure to salt stress. A high level of sugar and a delay in chlorophyll degradation were found in salt-tolerant rice. The total sugar content in leaf and root tissues of salt-tolerant rice was 2.47 and 2.85 times higher, respectively, than in the salt-sensitive variety. Meanwhile, less chlorophyll degradation was detected. Salt stress may promote sugar accumulation, thus preventing the degradation of chlorophyll. Water oxidation by the light reaction of photosynthesis in the salt-tolerant variety was greater than that in the salt-sensitive variety, indicated by a high maximum quantum yield of PSII (F _v/F _m) and quantum efficiency of PSII (Φ_PSII) with low nonphotochemical quenching (NPQ), leading to a high net photosynthetic rate. In addition, the overall growth performances in the salt-tolerant variety were higher than those in the salt-sensitive variety. The FBP gene expression and enzyme activity, sugar accumulation, pigment stabilization, water oxidation and net photosynthetic rate parameters in HJ rice should be further investigated as multivariate salt-tolerant indices for the classification of salt tolerance in rice breeding programs.     

capA, a cspA-like gene that encodes a cold acclimation protein in the psychrotrophic bacterium Arthrobacter globiformis SI55.

By use of Arthrobacter globiformis SI55, a psychrotrophic bacterium capable of growth between -5 and +32 degrees C, we cloned and sequenced capA, a gene homologous to cspA encoding the major cold shock protein in Escherichia coli. The deduced protein sequence has a high level of identity with the sequences of other CspA-related proteins from various sources, and no particular residue or domain that could be specific to cold-adapted microorganisms emerged. We show that CapA was produced very rapidly following cold shock, but unlike its mesophilic counterparts, it was still expressed during prolonged growth at low temperature. Its synthesis is regulated at the translational level, and we showed that growth resumption following a temperature downshift correlated with CapA expression. Transient inhibitions in protein synthesis during the first stages of the cold shock response severely impaired the subsequent acclimation of A. globiformis SI55 to low temperature and delayed CapA expression. The cold shock response in A. globiformis SI55 is an adaptative process in which CapA may play a crucial role. We suggest that low-temperature acclimation is conditioned mainly by the ability of cells to restore an active translational machinery after cold shock in a process that may be different from that present in mesophiles.     

Cloning, expression and characterization of trehalose-6-phosphate phosphatase from a psychrotrophic bacterium, Arthrobacter strain A3

A trehalose-6-phosphate phosphatase (TPP) gene, otsB, from a psychrotrophic bacterium, Arthrobacter strain A3, was identified. The product of this otsB gene is 266 amino acids in length with a calculated molecular weight of 27,873 Da. The protein was expressed in Escherichia coli and purified to apparent homogeneity. The purified recombinant TPP catalyzed the dephosphorylation of trehalose-6-phosphate to form trehalose and showed a broad optimum pH range from 5.0 to 7.5. This enzyme also showed an absolute requirement for Mg(2+) or Co(2+) for catalytic activity. The recombinant TPP had a maximum activity at 30 °C and maintained activity over a temperature range of 4-30 °C. TPP was generally heat-labile, losing 70 % of its activity when subjected to heat treatment at 50 °C for 6 min. Kinetic analysis of the Arthrobacter strain A3 TPP showed ~tenfold lower K (m) values when compared with values derived from other bacterial TPP enzymes. The highest k (cat)/K (m) value was 37.5 mM(-1) s(-1) (repeated three times), which is much higher than values published for mesophilic E. coli TPP, indicating that the Arthrobacter strain A3 TPP possessed excellent catalytic activity at low temperatures. Accordingly, these characteristics suggest that the TPP from the Arthrobacter strain A3 is a new cold-adapted enzyme. In addition, this is the first report characterizing the enzymatic properties of a TPP from a psychrotrophic organism.     

Salicylic acid-altering Arabidopsis mutants response to salt stress

Aims

The role of salicylic acid (SA) in plant responses to salinity is still a matter of controversy. To address the effect of endogenous SA variation in level and signaling on plant responses to salinity, biochemical and physiological analyses were performed on SA-altering Arabidopsis mutants including snc1 with high level of SA, transgenic line nahG with low SA, npr1-1 with SA signaling blockage, snc1/nahG plants (expression of nahG in the snc1 background), as well as wild type plants.

Methods

Plants were cultured in 1?×?Hoagland solution under controlled conditions. For salt exposure, NaCl at final concentrations of 100?mM, 200?mM, and 300?mM, respectively, was added to the culture solution after 25?d of seed germination. Except where mentioned, plant leaves were harvested after 14?d of salt stress, and used for physiological and chemical analyses.

Results

Salt stress caused all plants growth retardation with a dose-effect relationship relative to control. However, compared to wild type plants, a greater growth inhibition occurred in snc1, while a less inhibition was observed in nahG and npr1-1 plants, and a comparable extent was detected in snc1/nahG plants in which the SA level was near to that in wild type plants. The snc1 plants had lower net photosynthetic rate, variable to maximum fluorescence ratio, quantum efficiency of photosystem 2, reduced glutathione/oxidized glutathione ratio, proline levels, and higher malondiadehyde levels and electrolyte leakage rates as compared to wild type plants under salt stress. These values were effectively reversed by the expression of nahG gene in snc1 plants. The nahG and npr1-1 plants always exhibited more tolerance to salinity in above-mentioned indices than wild type plants. However, higher activities of superoxide dismutase and peroxidase in snc1 plants did not contribute to salt tolerance.

Conclusions

These data showed that SA deficit or signaling blockage in Arabidopsis plants was favorable to salt adaptation, while a high accumulation of SA potentiated salt-induced damage to Arabidopsis plants.     

Changes in the tobacco leaf apoplast proteome in response to salt stress

The apoplast of plant cells is a dynamic compartment involved in many processes, including maintenance of tissue shape, development, nutrition, signalling, detoxification and defence. In this work we used Nicotiana tabacum plants as a model to investigate changes in the soluble apoplast composition induced in response to salt stress. Apoplastic fluid was extracted from leaves of control plants and plants exposed to salt stress, using a vacuum infiltration procedure. Two-dimension electrophoretic analyses revealed about 150 polypeptide spots in the pH range of 3.0 to 10.0, in independent protein extracts, with a high level of reproducibility between the two sample sets. Quantitative evaluation and statistical analyses of the resolved spots in treated and untreated samples revealed 20 polypeptides whose abundance changed in response to salt stress. Mass spectroscopic peptide separation and sequencing was used to identify polypeptides affected by salt stress. While the levels of some proteins were reduced by salt-treatment, an enhanced accumulation of protein species known to be induced by biotic and abiotic stresses was observed. In particular, two chitinases and a germin-like protein increased significantly and two lipid transfer proteins were expressed entirely de novo. Some apoplastic polypeptides, involved in cell wall modifications during plant development, remained largely unchanged. The significance of these components is discussed in the context of stress responses in plants.     

Rapid increase of vacuolar volume in response to salt stress

Suspension-cultured cells of mangrove [Bruguiera sexangula (Lour.) Poir.] showed a rapid increase in vacuolar volume under salt stress, although there was no change in the cell volume. The rapid increase in the vacuolar volume was an active process, which followed the activation of the tonoplast H(+)-ATPase and the vacuolar acid phosphatase. The same phenomenon was observed in barley (Hordeum vulgare L. cv. Doriru) root meristematic cells under salt stress but not in pea ( Pisum sativum L.). Increases in vacuolar volume could potentially protect the cytoplasm by decreasing the cytoplasmic volume during the initial phases of salt stress.     

Adaptive changes of the yeast mitochondrial proteome in response to salt stress

Mitochondria are dynamic organelles with the capacity to adapt to environmental stimuli and stress. Here we use yeast (Saccharomyces cerevisiae) in combination with proteomic approaches to quantify the changes in the protein composition of mitochondria in the presence of salt stress provoked by NaCl. We identified 15 proteins that were more than twofold overrepresented in salt adapted mitochondria. These proteins are mainly involved in the oxidative stress defense, the biosynthesis of amino acids and ubiquinone or in the metabolism of pyruvate and acetate. Loss of function of most of the upregulated proteins did not result in a significant growth phenotype under high salt conditions. However, all identified proteins were necessary to sustain efficient growth under oxidative stress caused by hydrogen peroxide. Additionally, a subset of outer mitochondrial membrane proteins was shown to be upregulated upon salt stress. We furthermore identified nine proteins that were more than threefold underrepresented in salt adapted mitochondria. These proteins were mainly glycolytic enzymes or proteins with a predominant localization at the endoplasmatic reticulum. Our results underline the complex nature of the stress adaptation of mitochondria and identify functional groups of proteins whose specific role in salt resistance should be revealed in the future.     

Salicylic acid negatively affects the response to salt stress in pea plants

We studied the effect of salicylic acid (SA) treatment on the response of pea plants to salinity. Sodium chloride (NaCl)-induced damage to leaves was increased by SA, which was correlated with a reduction in plant growth. The content of reduced ascorbate and glutathione in leaves of salt-treated plants increased in response to SA, although accumulation of the respective oxidised forms occurred. An increase in hydrogen peroxide also occurred in leaves of salt-exposed plants treated with SA. In the absence of NaCl, SA increased ascorbate peroxidase (APX; 100 μm) and glutathione-S transferase (GST; 50 μm) activities and increased catalase (CAT) activity in a concentration-dependent manner. Salinity decreased glutathione reductase (GR) activity, but increased GST and CAT activity. In salt-stressed plants, SA also produced changes in antioxidative enzymes: 100 μm SA decreased APX but increased GST. Finally, a concentration-dependent increase in superoxide dismutase (SOD) activity was induced by SA treatment in salt-stressed plants. Induction of PR-1b was observed in NaCl-stressed plants treated with SA. The treatment with SA, as well as the interaction between salinity and SA treatment, had a significant effect on PsMAPK3 expression. The expression of PsMAPK3 was not altered by 70 mm NaCl, but was statistically higher in the absence than in the presence of SA. Overall, the results show that SA treatment negatively affected the response of pea plants to NaCl, and this response correlated with an imbalance in antioxidant metabolism. The data also show that SA treatment could enhance the resistance of salt-stressed plants to possible opportunistic pathogen attack, as suggested by increased PR-1b gene expression.     

The response of the filamentous cyanobacterium Spirulina platensis to salt stress

The responses of the filamentous cyanobacterium Spirulina platensis to increased NaCl concentrations (0.25–1.0 M) in addition to the concentration of sodium in the growth medium were studied. A two stage response to the salt stress was observed. This consisted of a relatively short shock stage, followed by adaptation process. It was shown that upon exposure to high salt concentrations of 0.5 M and above, immediate inhibition of photosynthesis and respiration, and complete cessation of growth occurred. After a time lag, the energy-yielding processes exhibited restored activity. At 0.5 and 1.0M NaCl photosynthesis reached 80% and 50% that of the control, while respiration was enhanced by 140 and 200%, respectively. The time lags were longer when the cells were exposed to higher NaCl concentrations. The resumption of growth and the establishment of new steady state growth rates were found to be correlated to the recovery in respiration. The relationship between the growth rates after adaptation and the increased NaCl concentrations was found to be inversely linear. The cellular sodium content was maintained at a constant low level, regardless of the external NaCl concentration, while potassium content declined linearly vs. the external NaCl concentration. The carbohydrate content of the cells rose exponentially with the increase in NaCl concentration.Publication No. 34 from the Micro-Algal Biotechnology Lab.     

The cold shock response of the psychrotrophic bacterium Pseudomonas fragi involves four low-molecular-mass nucleic acid-binding proteins.

The psychrotrophic bacterium Pseudomonas fragi was subjected to cold shocks from 30 or 20 to 5 degrees C. The downshifts were followed by a lag phase before growth resumed at a characteristic 5 degrees C growth rate. The analysis of protein patterns by two-dimentional gel electrophoresis revealed overexpression of 25 or 17 proteins and underexpression of 12 proteins following the 30- or 20-to-5 degrees C shift, respectively. The two downshifts shared similar variations of synthesis of 20 proteins. The kinetic analysis distinguished the induced proteins into cold shock proteins (Csps), which were rapidly but transiently overexpressed, and cold acclimation proteins (Caps), which were more or less rapidly induced but still overexpressed several hours after the downshifts. Among the cold-induced proteins, four low-molecular-mass proteins, two of them previously characterized as Caps (CapA and CapB), and heat acclimation proteins (Haps) as well as heat shock proteins (Hsps) for the two others (TapA and TapB) displayed higher levels of induction. Partial amino acid sequences, obtained by microsequencing, were used to design primers to amplify by PCR the four genes and then determine their nucleotide sequences. A BamHI-EcoRI restriction fragment of 1.9 kb, containing the complete coding sequence for capB, was cloned and sequenced. The four peptides belong to the family of small nucleic acid-binding proteins as CspA, the major Escherichia coli Csp. They are likely to play a major role in the adaptative response of P. fragi to environmental temperature changes.     

Formation of guaiacol in chocolate milk by the psychrotrophic bacterium Rahnella aquatilis

AIMS: The aim of this study was to identify the causative agent of a smoky/phenolic taint in refrigerated full cream chocolate milk. METHODS AND RESULTS: Microbiological examination of spoiled and unspoiled milk samples from the same processor showed high numbers of the psychrotrophic coliform Rahnella aquatilis in the spoiled samples only. Gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify the taint compound as guaiacol (2-methoxyphenol) in the spoiled milk. Challenge studies in UHT chocolate and white milks inoculated with the isolate and incubated at 4-5 degrees C and 8-9 degrees C for 6 d showed the production of guaiacol in chocolate milk only, which was confirmed and quantified by GC/MS. CONCLUSIONS: The results indicate that if present in refrigerated chocolate milk, Rah. aquatilis can produce guaiacol within the expected shelf-life of the product, even without temperature abuse. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that the coliform Rah. aquatilis can produce guaiacol in refrigerated chocolate milk products.     

Photosynthesis and yield traits in different soybean lines in response to salt stress

To evaluate utility of different salt-tolerant lines, three soybean lines with different resistance to salt were planted in the field under control and salt-stress conditions for two years. The results showed that net photosynthetic rate (P_N) was significantly different among lines at the anthesis stage and decreased on average by 13.6-34.1% under conditions of salt stress. The stomatal conductance was a primary limiting factor for the reduction of P_N under salt stress. Meanwhile, the grain yield (GY) decreased on average by 14.0-35.3% among lines under salt stress. The salt-tolerant lines S111-9 and S113-6 showed higher P_N and GY under salt stress in comparison with the salt-sensitive cultivar Melrose. Regression analysis indicated that there was extremely significantly positive correlation between GY and P_N under field conditions. Therefore, P_N might be used as a physiological index for field resistance of soybean to salt stress.     

Structural changes in root and shoot ofBacopa monniera in response to salt stress

Various concentrations of salt (NaCI) were shown to have an influence on the differentiation of tissues in the root and stem ofBacopa monniera (L) Wettst. Higher concentrations induced drastic changes in roots grown on salt-supplemented media; epidermal and cortical cells experienced changes in shape, size, and orientation and/or were got disintegrated. A low concentration of salt induced a profuse development of root hairs which gradually disappeared at higher concentrations. Air spaces in the stem cortex were enlarged and xylem cell walls in the vascular ring were thickened.