Differential Responses of Nitrogen-Fixing Cyanobacteria to Salinity and Osmotic Stresses

Two nitrogen-fixing Anabaena strains were found to be differentially tolerant to salinity and osmotic stresses. Anabaena torulosa, a brackish-water, salt-tolerant strain, was relatively osmosensitive. Anabaena sp. strain L-31, a freshwater, salt-sensitive strain, on the other hand, displayed significant osmotolerance. Salinity and osmotic stresses affected nitrogenase activity differently. Nitrogen fixation in both of the strains was severely inhibited by the ionic, but not by the osmotic, component of salinity stress. Such differential sensitivity of diazotrophy to salinity-osmotic stresses was observed irrespective of the inherent tolerance of the two strains to salt-osmotic stress. Exogenously added ammonium conferred significant protection against salinity stress but was ineffective against osmotic stress. Salinity and osmotic stresses also affected stress-induced gene expression differently. Synthesis of several proteins was repressed by salinity stress but not by equivalent or higher osmotic stress. Salinity and osmotic stresses induced many common proteins. In addition, unique salt stress- or osmotic stress-specific proteins were also induced in both strains, indicating differential regulation of protein synthesis by the two stresses. These data show that cyanobacterial sensitivity and responses to salinity and osmotic stresses are distinct, independent phenomena.     

Comparative analysis of proteins induced by heat shock, salinity, and osmotic stress in the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31.

Heat, salinity, or osmotic stress influenced protein synthesis in nitrogen-fixing Anabaena sp. strain L-31. Salinity and osmotic stresses were identical and specifically induced 15 polypeptides. Four polypeptides were unique to heat shock, and four other polypeptides were induced under every stress. The results demonstrate a commonality and a stress specificity of protein synthesis regulation.     

Proteomic screening of salt-stress-induced changes in plasma membranes of Synechocystis sp. strain PCC 6803

The plasma membrane of a cyanobacterial cell is crucial as barrier against the outer medium. It is also an energy-transducing membrane as well as essential for biogenesis of cyanobacterial photosystems and the endo-membrane system. Previously we have identified 57 different proteins in the plasma membrane of control cells from Synechocystis sp. strain PCC6803. In the present work, proteomic screening of salt-stress proteins in the plasma membrane resulted in identification of 109 proteins corresponding to 66 different gene products. Differential and quantitative analyses of 2-DE profiles of plasma membranes isolated from both control and salt-acclimated cells revealed that twenty proteins were enhanced/induced and five reduced during salt stress. More than half of the enhanced/induced proteins were periplasmic binding proteins of ABC-transporters or hypothetical proteins. Proteins that exhibited the highest enhancement during salt stress include FutA1 (Slr1295) and Vipp1 (Sll0617), which have been suggested to be involved in protection of photosystem II under iron deficiency and in thylakoid membrane formation, respectively. Other salt-stress proteins were regulatory proteins such as PII protein, LrtA, and a protein that belongs to CheY subfamily. The physiological significance of the identified salt-stress proteins in the plasma membrane is discussed integrating our current knowledge on cyanobacterial stress physiology.     

Heat-shock response and its contribution to thermotolerance of the nitrogen-fixing cyanobacterium<Emphasis Type="Italic"> Anabaena</Emphasis> sp. strain L-31

Compared to Escherichia coli, the nitrogen-fixing soil cyanobacterium Anabaena sp. strain L-31 exhibited significantly superior abilities to survive prolonged and continuous heat stress and recover therefrom. Temperature upshift induced the synthesis of heat-shock proteins of similar molecular mass in the two microbes. However, in Anabaena sp. strain L-31 the heat-shock proteins (particularly the GroEL proteins) were synthesised throughout the stress period, were much more stable and accumulated during heat stress. In contrast, in E. coli the heat-shock proteins were transiently synthesised, quickly turned over and did not accumulate. Nitrogenase activity of Anabaena cells of sp. strain L-31 continuously exposed to heat stress for 7 days rapidly recovered from thermal injury, although growth recovery was delayed. Exposure of E. coli cells to >4.5 h of heat stress resulted in a complete loss of viability and the ability to recover. Marked differences in the synthesis, stability and accumulation of heat-shock proteins appear to distinguish these bacteria in their thermotolerance and recovery from heat stress.     

Salinity stress induced tissue-specific proteins in barley seedlings

Protein changes induced by salinity stress were investigated in two barley cultivars, California Mariout, a salt-tolerant variety and Prato, a salt-sensitive variety. Rapidly growing young barley seedlings were exposed to NaCl and the newly synthesized proteins were resolved on two dimensional polyacrylamide gels following isoelectric focusing or nonequilibrium pH gradient gel electrophoresis in the first dimension. Salinity induces distinct protein changes in root and shoot tissues. In roots, the salinity effects are identical in both cultivars. First, salinity modulates the synthesis of two different sets of proteins, one of which is elevated, and the other, depressed. Second, six new proteins are induced all of which are low in molecular weight, 24 to 27 kilodaltons, with an isoelectric point range of 6.1 to 7.6. In contrast to roots, salinity induces cultivar-specific shoot proteins. Five new shoot proteins are induced whose molecular weights and isoelectric points fall within the range of 20 to 24 kilodaltons and 6.3 to 7.2, respectively. Three of the newly induced proteins are unique to Prato. In addition, salinity inhibits the synthesis of a majority of shoot proteins. The new proteins produced in roots and shoots are unique to each tissue and their induction is apparently regulated coordinately during salinity stress.     

Insight into the salt tolerance factors of a wild halophytic rice, <Emphasis Type="Italic">Porteresia coarctata</Emphasis>: a physiological and proteomic approach

Salinity poses a serious threat to yield performance of cultivated rice in South Asian countries. To understand the mechanism of salt-tolerance of the wild halophytic rice, Porteresia coarctata in contrast to the salt-sensitive domesticated rice Oryza sativa, we have compared P. coarctata with the domesticated O. sativa rice varieties under salinity stress with respect to several physiological parameters and changes in leaf protein expression. P. coarctata showed a better growth performance and biomass under salinity stress. Relative water content was conserved in Porteresia during stress and sodium ion accumulation in leaves was comparatively lesser. Scanning electron microscopy revealed presence of two types of salt hairs on two leaf surfaces, each showing a different behaviour under stress. High salt stress for prolonged period also revealed accumulation of extruded NaCl crystals on leaf surface. Changes induced in leaf proteins were studied by two-dimensional gel electrophoresis and subsequent quantitative image analysis. Out of more than 700 protein spots reproducibly detected and analyzed, 60% spots showed significant changes under salinity. Many proteins showed steady patterns of up- or downregulation in response to salinity stress. Twenty protein spots were analyzed by MALDI-TOF, leading to identification of 16 proteins involved in osmolyte synthesis, photosystem functioning, RubisCO activation, cell wall synthesis and chaperone functions. We hypothesize that some of these proteins confer a physiological advantage on Porteresia under salinity, and suggest a pattern of salt tolerance strategies operative in salt-marsh grasses. In addition, such proteins may turn out to be potential targets for recombinant cloning and introgression in salt-sensitive plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Antioxidant response and proteomic modulations in Indian mustard grown under salt stress

Productivity of Indian mustard (Brassica juncea L. Czern. and Coss.) is markedly reduced by salt stress. To develop salt tolerance in this important oilseed crop is a need of the hour. This study, based on analysis of growth parameters and antioxidant profile of fourteen Indian mustard genotypes treated with 50, 100, 150 and 200 mM of sodium chloride, was performed to identify the salt-sensitive and salt-tolerant genotypes. Salinity stress inhibited biomass accumulation and reduced the protein and chlorophyll contents in a dose-dependent manner. The reduction was the highest in genotype Pusa Agrani and lowest in CS-54, depicting their contrasting sensitivity to salt stress. Salt treatments triggered a concentration-dependent overproduction of reactive-oxygen species and a concurrent upregulation of the expression of different antioxidants. Genotype CS-54 showed the least damage and maintained a high antioxidant level with almost each salt treatment, exhibiting its competence to withstand the damage provoked by salinity stress. Genotype Pusa Agrani, on the contrary, depicted a salt-sensitive nature by way of its very high lipid peroxidation and low intensity of antioxidants. These two genotypes were further investigated through gel-based proteomic approach, which resulted in the identification and quantification of 42 salinity-responsive proteins related to different metabolic modifications. Molecular processes, including photosynthesis, redox homeostasis, nitrogen metabolism, ATP synthesis, protein synthesis and degradation, signal transduction and respiratory pathways, have exhibited significant changes. The identified stress-responsive proteins could pave the way to develop salt tolerance in Indian mustard plant, thus sustaining its productivity under salinity.     

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.     

Possible amelioration of coastal soil salinity using halotolerant nitrogen-fixing cyanobacteria

A brackish-water, nitrogen-fixing cyanobacterium, Anabaena torulosa, could successfully grow and fix nitrogen on moderately saline Kharland soils (soil conductivity 5 to 8.50 dS m-1), typical of Indian coastline. During five weeks of growth under laboratory as well as field conditions, the cyanobacterium exhibited high rates of nitrogen fixation and substantially enriched the nitrogen status of saline soils (43-76%), although the fixed nitrogen remained confined to the cyanobacterial biomass. Most (>90%) of the cell-bound Na+ remained extracellularly trapped in the mucopolysaccharide sheath of A. torulosa; traces of the cation that permeated cyanobacterial cells were found to exist in an osmotically active, free state. No evidence was found for the incorporation of Na+ into any biomolecule, especialty proteins or carbohydrates. Therefore, permanent removal of Na+ from saline soils using cyanobacteria may not be possible, since Na+ is released back into the soil subsequent to the death and decay of cyanobacteria. Removal of top soil containing cyanobacterial mats significantly decreased the soil salinity (between 26-38%). But such a practice removes all the fixed nitrogen and carbon and also does not seem feasible on a large scale. Amelioration of soil salinity by simultaneous application of A. torulosa during crop growth seems to be an attractive possibility, especially since it can also supplement the nitrogen requirement of the crop.     

Proteomic identification of OsCYP2, a rice cyclophilin that confers salt tolerance in rice (Oryza sativa L.) seedlings when overexpressed

Background  

High Salinity is a major environmental stress influencing growth and development of rice. Comparative proteomic analysis of hybrid rice shoot proteins from Shanyou 10 seedlings, a salt-tolerant hybrid variety, and Liangyoupeijiu seedlings, a salt-sensitive hybrid variety, was performed to identify new components involved in salt-stress signaling.     

PGPR regulate caspase-like activity,programmed cell death,and antioxidant enzyme activity in paddy under salinity

The response of two root associated bacteria Pseudomonas pseudoalcaligenes and Bacillus pumilus were studied in the (salt-sensitive) rice GJ17 cultivar to salinity under controlled environmental growth conditions for protection of plant from adverse effect of salinity. Salinity affects the growth of salt-sensitive cultivar, but inoculation of plant growth promoting rhizobacteria (PGPR) reduces the harmful effect of salinity. The present study states that PGPR helps to reduce lipid peroxidation and superoxide dismutase activity in salt-sensitive GJ17 cultivar under salinity and play an important role in the growth regulation for positive adaptation of plants to salt stress. This study shows that inoculation of paddy (Oryza sativa) with such bacteria could provide salt-tolerant ability by reducing the toxicity of reactive oxygen species by reducing plant cell membrane index, cell caspase-like protease activity, and programmed cell death and hence resulted in increase cell viability. As these isolates remain associated with the roots, the effects of tolerance against salinity are observed here. Results also indicate that isolated PGPR strain help in alleviating up to 1.5 % salinity stress as well as improve tolerance.     

Regulation of Potassium-Dependent Kdp-ATPase Expression in the Nitrogen-Fixing Cyanobacterium Anabaena torulosa

The KdpB polypeptides in the cyanobacterium Anabaena torulosa were shown to be two membrane-bound proteins of about 78 kDa, expressed strictly under K(+) deficiency and repressed or degraded upon readdition of K(+). In both Anabaena and Escherichia coli strain MC4100, osmotic and ionic stresses caused no significant induction of steady-state KdpB levels during extreme potassium starvation.     

Salinity effects on growth,photosynthetic parameters,and nitrogenase activity in estuarine planktonic cyanobacteria

Salinity has been suggested as being a controlling factor for blooms of N2-fixing cyanobacteria in estuaries. We tested the effect of salinity on the growth, N2 fixation, and photosynthetic activities of estuarine and freshwater isolates of heterocystous bloom-forming cyanobacteria. Anabaena aphanizomenoides and Anabaenopsis sp. were isolated from the Neuse River Estuary, North Carolina, and Cylindrospermopsis raciborskii from Lakes Dora and Griffin, central Florida. Salinity tolerance of these cyanobacteria was compared with that of two Nodularia strains from the Baltic Sea. We measured growth rates, N2 fixation (nitrogenase activity), and CO2 fixation at salinities between 0 and 20 g L(-1) NaCl. We also examined photosynthesis-irradiance relation-ships in response to salinity. Anabaenopsis maintained similar growth rates in the full range of salinities from 2 to 20 g L(-1) NaCl. Anabaena grew at up to 15 g L-', but the maximum salinity 20 g L(-1) NaCl was inhibitory. The upper limit for salinity tolerance of Cylindrospermopsis was 4 g L(-1) NaCl. Nodularia spp. maintained similar growth rates in the full range of salinities from 0 to 20 g L(-1) . Between 0 and 10 g L(-1), the growth rate of Nodularia spumigena was slower than that of the Neuse Estuary strains. In most strains, the sensitivity of nitrogenase activity and CO2 fixation to salinity appeared similar. Anabaenopsis, Anabaena, and the two Nodularia strains rapidly responded to NaCl by increasing their maximum photosynthetic rates (Pmn). Overall, both Neuse River Estuary and Baltic Sea strains showed an ability to acclimate to salt stress over short-(24 h) and long-term (several days to weeks) exposures. The study suggested that direct effect of salinity (as NaCl in these experiments) on cyanobacterial physiology does not alone explain the low frequency and magnitude of blooms of N2-fixing cyanobacteria in estuaries.     

Protein profiling with cleavable isotope-coded affinity tag (cICAT) reagents: the yeast salinity stress response

Protein expression profiles in yeast cells, in response to salinity stress, were determined using the cleavable isotope-coded affinity tag (cICAT) labeling strategy. The analysis included separation of the mixed protein samples by SDS-PAGE, followed by excision of the entire gel lane, and division of the lane into 14 gel regions. Regions were subjected to in-gel digestion, biotin affinity chromatography, and analysis by nano-scale microcapillary liquid chromatography coupled to tandem mass spectrometry. The novel (13)C-labeled ICAT reagents have identical elution profiles for labeled peptide pairs and broadly spread the distribution of labeled peptides during reversed-phase chromatography. A total of 560 proteins were identified and quantified, with 51 displaying more than 2-fold expression differences. In addition to some known proteins involved in salt stress, four RNA-binding proteins were found to be up-regulated by high salinity, suggesting that selective RNA export from the nucleus is important for the salt-stress response. Some proteins involved in amino acid synthesis, which have been observed to be up-regulated by amino acid starvation, were also found to increase their abundance on salt stress. These results indicate that salt stress and amino acid starvation cause overlapping cellular responses and are likely to be physiologically linked.     

Proteomic Analysis of Seedling Roots of Two Maize Inbred Lines That Differ Significantly in the Salt Stress Response

Salinity is a major abiotic stress that limits plant productivity and quality throughout the world. Roots are the sites of salt uptake. To better understand salt stress responses in maize, we performed a comparative proteomic analysis of seedling roots from the salt-tolerant genotype F63 and the salt-sensitive genotype F35 under 160 mM NaCl treatment for 2 days. Under salinity conditions, the shoot fresh weight and relative water content were significantly higher in F63 than in F35, while the osmotic potential was significantly lower and the reduction of the K⁺/Na⁺ ratio was significantly less pronounced in F63 than in F35. Using an iTRAQ approach, twenty-eight proteins showed more than 2.0- fold changes in abundance and were regarded as salt-responsive proteins. Among them, twenty-two were specifically regulated in F63 but remained constant in F35. These proteins were mainly involved in signal processing, water conservation, protein synthesis and biotic cross-tolerance, and could be the major contributors to the tolerant genotype of F63. Functional analysis of a salt-responsive protein was performed in yeast as a case study to confirm the salt-related functions of detected proteins. Taken together, the results of this study may be helpful for further elucidating salt tolerance mechanisms in maize.     

Salt tolerance-related protein STO binds to a Myb transcription factor homologue and confers salt tolerance in Arabidopsis

Regulating the intracellular Na+/K+ ratio is an essential process for salinity tolerance. The yeast mutant, can, which is deficient in calcineurin, can not grow on medium containing Na+ because it is unable to regulate the intracellular Na+/K+ ratio. Expression of the STO gene of Arabidopsis thaliana in the can mutant complements the salt-sensitive phenotype. A protein of Arabidopsis, an H-protein promoter binding factor (HPPBF-1), that binds to STO protein was isolated. HPPBF-1 cDNA has a sequence encoding a Myb DNA binding-motif and its gene expression is induced by salt stress. Furthermore, HPPBF-1 protein is localized in the nucleus. Although, the expression level of STO is not induced under salt-stress conditions, overexpression of STO in a transgenic Arabidopsis plant gave it a higher salt tolerance than was observed in the wild type. When STO transgenic plants and wild-type plants were subjected to salt stress, root growth was increased by 33-70% in the transgenic plants under salt stress. These results suggest that STO is involved in salt-stress responses in Arabidopsis.     

Osmoregulation in Dunaliella, Part I: Effects of osmotic stress on photosynthesis, dark respiration and glycerol metabolism in Dunaliella tertiolecta and its salt-sensitive mutant (HL 25/8).

The photosynthetic oxygen evolution increased by about 30% over control when Dunaliella tertiolecta and its salt-sensitive mutant (HL 25/8) were stressed by raising NaCl concentration from 0.17 to 0.4M, however, during the dilution stress the photosynthetic oxygen evolution was progressively decreased with increasing dilution (decreasing the salinity). The photosynthetic oxygen evolution is affected by the water potential of the medium rather than by the ionic strength. Mitochondrial dark respiration was unaffected by salt stress, however, it was increased by about 50% (parent strain) and 35% (the mutant) upon dilution as if reduced pyridine nucleotide generated during glycerol dissimilation reaction were continuously oxidized by the mitochondria. The salt stress-induced changes in photosynthetic (14)CO(2) fixation were consistent with the observed rates of photosynthetic oxygen evolution. The mutant strain showed about one-half the capability for photosynthesis, and glycerol synthesis compared to the parent strain. However, the proportion of photosynthetically newly fixed carbon during salt stress in glycerol was similar in both strains. The glycerol dissimilation capabilities of both strains were also similar. It is suggested that the salt sensitivity of the mutant is probably due to its reduced dissolved inorganic carbon transport, photosynthetic and starch metabolism capabilities to provide carbon for glycerol synthesis in the time frame of adaptation process.