Salt-stressed Cells Research Articles

Involvement of GLR-mediated nitric oxide effects on ROS metabolism in Arabidopsis plants under salt stress

Plant glutamate receptor-like channels (GLRs) play important roles in plant development, immune response, defense signaling and Nitric oxide (NO) production. However, their involvement in abiotic stress responses, particularly in regulating Reactive Oxygen Species (ROS), is not well understood. This study aimed to investigate GLR-mediated NO production on ROS regulation in salt-stressed cells. To achieve this, Arabidopsis thaliana Columbia (Col-0) were treated with NaCl, glutamate antagonists [(DNQX (6,7-dinitroquinoxaline-2,3-dione and AP-5(D-2-amino-5-phosphono pentanoic acid)], and NO scavenger [cPTIO (2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt)]. Salt-stressed plants in combination with DNQX and AP-5 have exhibited higher increase in lipid peroxidation (TBARS), hydrogen peroxide (H2O2) and superoxide radical (O−2) contents as compared to solely NaCl-treated plants. Furthermore, NO and total glutathione contents, and S-nitrosoglutathione reductase (GSNOR) activity decreased with these treatments. AP-5 and DNQX increased the activities of NADPH oxidase (NOX), catalase (CAT), peroxidase (POX), cell wall peroxidase (CWPOX) in salt-stressed Arabidopsis leaves. However, their activities (except NOX) were significantly inhibited by cPTIO. Conversely, the combination of NaCl and GLR antagonists, NO scavenger decreased the activities of ascorbate peroxidase (APX), superoxide dismutase (SOD), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) resulting in elevated GSSG levels, a low GSH/GSSG ratio, impaired ROS scavenging, excessive ROS accumulation and cell membrane damage. The findings of this study provide evidence that GLR-mediated NO plays a crucial role in improvement of the tolerance of Arabidopsis plants to salt-induced oxidative stress. It helps to maintain cellular redox homeostasis by reducing ROS accumulation and increasing the activity of SOD, GSNOR, and the ASC-GSH cycle enzymes.

Ion fluxes Involved in the Adaptation of the Estuarine Diatom Coscinodiscus centralis Ehrenberg to Salinity Stress.

Although estuarine diatoms have a wide range of salt tolerance, they are often severely stressed by elevated salt concentrations. It remains poorly understood how estuarine diatoms maintain ionic homeostasis under high-salinity conditions. Using a scanning ion-selective electrode technique, this study determined the fluxes of H+, Na+, and K+ involved in the acclimatization of the estuarine diatom Coscinodiscus centralis Ehrenberg after an elevation in salinity from 15 psu to 35 psu. The C. centralis cells exhibited marked H+ effluxes after a transient treatment (TT, 30 min) and short-term treatment (ST, 24 h). However, a drastic shift of H+ efflux toward an influx was induced in the long-term treatment (LT, 10 days). The Na+ flux under TT, ST, and LT salinity conditions was found to accelerate the Na+ efflux. More pronounced effects were observed under the ST and LT salinity conditions compared to the TT salinity condition. The K+ influx showed a significant increase under the LT salinity condition. However, the salinity-induced Na+/H+ exchange in the estuarine diatom was inhibited by amiloride and sodium orthovanadate. These results indicate that the Na+ extrusion in salt-stressed cells is mainly the result of an active Na+/H+ antiport across the plasma membrane. The pattern of ion fluxes under the TT and ST salinity conditions were different from those under the LT salinity conditions, suggesting an incomplete regulation of the acclimation process in the estuarine diatom under short-term salinity stress.

Exogenous melatonin improves salt tolerance mainly by regulating the antioxidant system in cyanobacterium Nostoc flagelliforme.

Melatonin is a multifunctional nontoxic bio-stimulant or signaling molecule, generally distributing in different animal and plant organs for invigorating numerous physiological processes against abiotic stresses. In this study, we investigated the potential impact of melatonin on the cyanobacterium Nostoc flagelliforme when exposed to salt stress according to some biochemical and physiological parameters, such as relative electrolyte leakage, PSII activity, and photosynthetic pigments including chlorophyll a, phycocyanobilin, and phycoerythrobilin. We found that melatonin could also maintain K+ homeostasis in salt-stressed N. flagelliforme. These above results confirmed melatonin had multiple functions in hyperosmotic stress and ion stress caused by salinity. Notably, we observed melatonin could regulate the reactive oxygen species (ROS) signal and distinctly decrease the content of hydrogen peroxide and superoxide anion in salt-stressed cells, which were largely attributed to the increased antioxidant enzymes activities including catalase, superoxide dismutase, ascorbate peroxidase, and glutathione reductase. Finally, qRT-PCR analysis showed that melatonin stimulated the expression of antioxidant genes (NfCAT, NfSOD, and NfGR). In general, our findings demonstrate melatonin has beneficial effects on N. flagelliforme under salt stress by intensively regulating antioxidant system.

Adaptation to Varying Salinity in Halomonas elongata: Much More Than Ectoine Accumulation.

The halophilic γ-proteobacterium Halomonas elongata DSM 2581T thrives at salt concentrations well above 10 % NaCl (1.7 M NaCl). A well-known osmoregulatory mechanism is the accumulation of the compatible solute ectoine within the cell in response to osmotic stress. While ectoine accumulation is central to osmoregulation and promotes resistance to high salinity in halophilic bacteria, ectoine has this effect only to a much lesser extent in non-halophiles. We carried out transcriptome analysis of H. elongata grown on two different carbon sources (acetate or glucose), and low (0.17 M NaCl), medium (1 M), and high salinity (2 M) to identify additional mechanisms for adaptation to high saline environments. To avoid a methodological bias, the transcripts were evaluated by applying two methods, DESeq2 and Transcripts Per Million (TPM). The differentially transcribed genes in response to the available carbon sources and salt stress were then compared to the transcriptome profile of Chromohalobacter salexigens, a closely related moderate halophilic bacterium. Transcriptome profiling supports the notion that glucose is degraded via the cytoplasmic Entner-Doudoroff pathway, whereas the Embden-Meyerhoff-Parnas pathway is employed for gluconeogenesis. The machinery of oxidative phosphorylation in H. elongata and C. salexigens differs greatly from that of non-halophilic organisms, and electron flow can occur from quinone to oxygen along four alternative routes. Two of these pathways via cytochrome bo' and cytochrome bd quinol oxidases seem to be upregulated in salt stressed cells. Among the most highly regulated genes in H. elongata and C. salexigens are those encoding chemotaxis and motility proteins, with genes for chemotaxis and flagellar assembly severely downregulated at low salt concentrations. We also compared transcripts at low and high-salt stress (low growth rate) with transcripts at optimal salt concentration and found that the majority of regulated genes were down-regulated in stressed cells, including many genes involved in carbohydrate metabolism, while ribosome synthesis was up-regulated, which is in contrast to what is known from non-halophiles at slow growth. Finally, comparing the acidity of the cytoplasmic proteomes of non-halophiles, extreme halophiles and moderate halophiles suggests adaptation to an increased cytoplasmic ion concentration of H. elongata. Taken together, these results lead us to propose a model for salt tolerance in H. elongata where ion accumulation plays a greater role in salt tolerance than previously assumed.

Effect of salt stress on physiological parameters of microalgae Vischeria punctata strain IPPAS H-242, a superproducer of eicosapentaenoic acid

The strain IPPAS H-242 is an eustigmatophycean alga with good growth characteristics and high content of the long chain polyunsaturated eicosapentaenoic fatty acid (EPA) – a very-long-chain fatty acid with high nutraceutical value. In this study, based on 18S rRNA gene and ITS1–5.8S–ITS2 sequences the strain IPPAS H-242 was identified as an authentic strain of Vischeria punctata. The effect of salt stress (0.5 M NaCl) on growth, cell morphology, ultrastructure, and biochemical composition with the emphasis on the fatty acid (FA) profile was investigated in batch cultures. Under salt stress, biomass accumulation and cell division were severely inhibited; cells were bigger, with higher chloroplast volume and numerous mitochondria, they had more proteins (73 % from the initial concentration as compared to 23 % in control) and their lipids had higher EPA proportion (13.6 % of total FA as compared to 6.4 % of total FA in control). In salt-stressed cells, thylakoid organization and photosynthetic activity were impaired, and D1 protein content decreased to trace amounts. In spite of an increase in EPA proportion in total FA, salt stress causes a decrease in total EPA productivity (49 mg/L as compared to 130 mg/L in control).

The novel strain Desmonostoc salinum CCM-UFV059 shows higher salt and desiccation resistance compared to the model strain Nostoc sp. PCC7120.

Desmonostoc salinum CCM-UFV059 (Desmonostoc) is a novel cyanobacterial strain of the order Nostocales isolated from a saline-alkaline lake. The acclimation towards salt and desiccation stress of Desmonostoc was compared to the related and well-characterized model strain Nostoc sp. PCC7120 (Nostoc). Salt-stressed cells of Desmonostoc maintained low cellular Na+ concentrations and accumulated high amounts of compatible solutes, mainly sucrose and to a lower extent trehalose. These features permitted Desmonostoc to grow and maintain photosynthesis at 2-fold higher salinities than Nostoc. Moreover, Desmonostoc also induced sucrose over-accumulation under desiccation, which allowed this strain to recover from this stress in contrast to Nostoc. Additional mechanisms such as the presence of highly unsaturated lipids in the membrane and an efficient ion transport system could also explain, at least partially, how Desmonostoc is able to acclimate to high salinities and to resist longer desiccation periods. Collectively, our results provide first insights into the physiological and metabolic adaptations explaining the remarkable high salt and desiccation tolerance, which qualify Desmonostoc as an attractive model for further analysis of stress acclimation among heterocystous N2 -fixing cyanobacteria.

Three-dimensional ultrastructural change of chloroplasts in rice mesophyll cells responding to salt stress.

Excess salinity inhibits the metabolism of various systems and induces structural changes, especially in chloroplasts. Although the chloroplast body seems to swell under salinity stress as observed by conventional transmission electron microscopy, previous studies are limited to 2-D data and lack quantitative comparisons because specimens need to be sliced into ultrathin sections. This study shows three-dimensionally the structural changes in a whole mesophyll cell responding to salinity stress by serial sectioning with a focused ion beam scanning electron microscope (FIB-SEM) and compares the differences in chloroplast structures based on reconstructed models possessing accurate numerical voxel values. Leaf blades of rice plants treated with 100 mm NaCl or without (control) for 4 d were fixed chemically and embedded in resin. The specimen blocks were sectioned and observed using the FIB-SEM, and then the sliced image stacks were reconstructed into 3-D models by image processing software. On the transverse sections of rice mesophyll cells, the chloroplasts in the control leaves appeared to be elongated meniscus lens shaped, while those in the salt-treated leaves appear to be expanded oval shaped. The 3-D models based on serial sectioning images showed that the chloroplasts in the control cells spread like sheets fitted to the shape of the cell wall and in close contact with the adjacent chloroplasts. In contrast, those in the salt-stressed cells curled up into a ball and fitted to cell protuberances without being in close contact with adjacent chloroplasts. Although the shapes of chloroplasts were clearly different between the two treatments, their volumes did not differ. The 3-D reconstructed models of whole rice mesophyll cells indicated that chloroplasts under salt stress conditions were not swollen but became spherical without increasing their volume. This is in contrast to findings of previous studies based on 2-D images.

YocM a small heat shock protein can protect Bacillus subtilis cells during salt stress.

Small heat shock proteins (sHsp) occur in all domains of life. By interacting with misfolded or aggregated proteins these chaperones fulfill a protective role in cellular protein homeostasis. Here, we demonstrate that the sHsp YocM of the Gram-positive model organism Bacillussubtilis is part of the cellular protein quality control system with a specific role in salt stress response. In the absence of YocM the survival of salt shocked cells is impaired, and increased levels of YocM protect B. subtilis exposed to heat or salt. We observed a salt and heat stress-induced localization of YocM to intracellular protein aggregates. Interestingly, purified YocM appears to accelerate protein aggregation of different model substrates in vitro. In addition, the combined presence of YocM and chemical chaperones, which accumulate in salt stressed cells, can facilitate in vitro a synergistic protective effect on protein misfolding. Therefore, the beneficial role of YocM during salt stress could be related to a mutual functional relationship with chemical chaperones and adds a new possible functional aspect to sHsp chaperone activities.

Compatible Solute Synthesis and Import by the Moderate Halophile Spiribacter salinus: Physiology and Genomics.

Members of the genus Spiribacter are found worldwide and are abundant in ecosystems possessing intermediate salinities between seawater and saturated salt concentrations. Spiribacter salinus M19-40 is the type species of this genus and its first cultivated representative. In the habitats of S. salinus M19-40, high salinity is a key determinant for growth and we therefore focused on the cellular adjustment strategy to this persistent environmental challenge. We coupled these experimental studies to the in silico mining of the genome sequence of this moderate halophile with respect to systems allowing this bacterium to control its potassium and sodium pools, and its ability to import and synthesize compatible solutes. S. salinus M19-40 produces enhanced levels of the compatible solute ectoine, both under optimal and growth-challenging salt concentrations, but the genes encoding the corresponding biosynthetic enzymes are not organized in a canonical ectABC operon. Instead, they are scrambled (ectAC; ectB) and are physically separated from each other on the S. salinus M19-40 genome. Genomes of many phylogenetically related bacteria also exhibit a non-canonical organization of the ect genes. S. salinus M19-40 also synthesizes trehalose, but this compatible solute seems to make only a minor contribution to the cytoplasmic solute pool under osmotic stress conditions. However, its cellular levels increase substantially in stationary phase cells grown under optimal salt concentrations. In silico genome mining revealed that S. salinus M19-40 possesses different types of uptake systems for compatible solutes. Among the set of compatible solutes tested in an osmostress protection growth assay, glycine betaine and arsenobetaine were the most effective. Transport studies with radiolabeled glycine betaine showed that S. salinus M19-40 increases the pool size of this osmolyte in a fashion that is sensitively tied to the prevalent salinity of the growth medium. It was amassed in salt-stressed cells in unmodified form and suppressed the synthesis of ectoine. In conclusion, the data presented here allow us to derive a genome-scale picture of the cellular adjustment strategy of a species that represents an environmentally abundant group of ecophysiologically important halophilic microorganisms.

Physiological responses to salt stress of salt-adapted and directly salt (NaCl and NaCl+Na2SO4 mixture)-stressed cyanobacterium Anabaena fertilissima.

Soil salinity in nature is generally mixed type; however, most of the studies on salt toxicity are performed with NaCl and little is known about sulfur type of salinity (Na2SO4). Present study discerns the physiologic mechanisms responsible for salt tolerance in salt-adapted Anabaena fertilissima, and responses of directly stressed parent cells to NaCl and NaCl+Na2SO4 mixture. NaCl at 500mM was lethal to the cyanobacterium, whereas salt-adapted cells grew luxuriantly. Salinity impaired gross photosynthesis, electron transport activities, and respiration in parent cells, but not in the salt-adapted cells, except a marginal increase in PSI activity. Despite higher Na+ concentration in the salt mixture, equimolar NaCl appeared more inhibitive to growth. Sucrose and trehalose content and antioxidant activities were maximal in 250mM NaCl-treated cells, followed by salt mixture and was almost identical in salt-adapted (exposed to 500mm NaCl) and control cells, except a marginal increase in ascorbate peroxidase activity and an additional fourth superoxide dismutase isoform. Catalase isoform of 63kDa was induced only in salt-stressed cells. Salinity increased the uptake of intracellular Na+ and Ca2+ and leakage of K+ in parent cells, while cation level in salt-adapted cells was comparable to control. Though there was differential increase in intracellular Ca2+ under different salt treatments, ratio of Ca2+/Na+ remained the same. It is inferred that stepwise increment in the salt concentration enabled the cyanobacterium to undergo priming effect and acquire robust and efficient defense system involving the least energy.

Effects of Reduced and Enhanced Glycogen Pools on Salt-Induced Sucrose Production in a Sucrose-Secreting Strain of Synechococcus elongatus PCC 7942.

Sucrose and glycogen syntheses in cyanobacteria share the common precursor glucose-1-phosphate. It is generally assumed that lowering glycogen synthesis could drive more carbon toward sucrose synthesis that can be induced by salt stress among cyanobacteria. By using a theophylline-dependent riboswitch system, the expression of glgC, a key gene in glycogen synthesis, was downregulated in a quantitative manner in a sucrose-secreting strain of Synechococcus elongatus PCC 7942. We observed that the stepwise suppression of glycogen synthesis limited rather than stimulated sucrose production in the salt-stressed cells, suggesting that glycogen could serve as a carbon pool for the synthesis of sucrose. Accordingly, we generated glycogen-overproducing strains, but the increased glycogen pool alone did not stimulate sucrose production, indicating that alternative steps limit the carbon flux toward the synthesis of sucrose. Consistent with previous studies that showed that sucrose-phosphate synthase (SPS) catalyzes the rate-limiting step in sucrose synthesis, the combination of glycogen overproduction and sps overexpression resulted in increased sucrose production. Our results indicate that the glycogen and sucrose pools are closely linked in Synechococcus elongatus PCC 7942, and we propose that enhancing the glycogen pool could be a promising strategy for the improvement of sucrose production by cyanobacteria in the presence of a strong sucrose synthesis sink.IMPORTANCE Many cyanobacteria naturally synthesize and accumulate sucrose when stressed by NaCl, which provides novel possibilities for obtaining sugar feedstock by engineering of cyanobacteria. It has been assumed that glycogen synthesis competes with sucrose synthesis for the carbon flux. However, our results showed that the suppression of glycogen synthesis decreased rather than stimulated sucrose production in a sucrose-secreting strain of Synechococcus elongatus PCC 7942. This result suggests that glycogen could serve as a supportive rather than a competitive carbon pool for the synthesis of sucrose, providing new insights about the relation between glycogen synthesis and sucrose synthesis in cyanobacteria. This finding is also useful to guide metabolic engineering work to optimize the production of sucrose and possibly other products by cyanobacteria.

Pleiotropic Effect of Salt Stress on Motility and Synthesis of Secreted Ribonucleases by Bacillus pumilus

Members of the genus Bacillus can successfully counteract a sudden increase in salinity. In addition to the accumulation of osmolytes, saline stress also affects other aspects of bacterial physiology such as exoenzymes synthesis and motility. Here, we have shown that increase of salinity in growth medium leads to elevated biosynthesis level of low-molecular weight ribonuclease (RNase) binase I from Bacillus pumilus. The same effect was established previously for high-molecular weight binase II. Transmission electron microscopy revealed the absence of flagella and some other changes in salt-stressed cells of B. pumilis. We also detected the gene sequences homologous to the recognition sites of response regulator DegU in the binase I and binase II promoters. Using the B. subtilis strains with various mutations in DegU gene, we found that the two-component signal transduction system DegS-DegU which regulates the motility under salt stress participates in the control of biosynthesis for both secreted RNase of B. pumilis (binase I and binase II).

Physiological Basis for the Tolerance of Yeast Zygosaccharomyces bisporus to Salt Stress

Zygosaccharomyces bisporus is a moderately halotolerant yeast isolated from highly sugary and salty foods. We performed various evident biochemical and in vivo experiments as first of its kind to sketch out the possible overlay of salt tolerance mechanism in this model organism. The growth and survival curve analysis revealed that 1.0 M NaCl concentration (sublethal) enacts growth inhibitory effects with prompting immediate delay in cell division cycle; however, yeast cells adopted modified stress physiologically with further stretched stress spans which was accompanied by an upsurge in the level of cellular metabolites such as trehalose (reserve carbohydrate) and chiefly glycerol (polyols) as major compatible osmolytes, suggesting their role in defense mechanism against osmotic stress. To further elucidate the relation of osmotic stress cell physiology to salinity, thiobarbituric acid reactive substances, protein carbonyl, and reduced glutathione content were measured in salt-stressed cells demonstrating positive correlation of reactive oxygen species generation in Z. bisporus with an elevated concentration of lipid and protein oxidation, thereby damaging cell membrane and eventually causing cell death. We assessed NaCl exposure sourcing increased intracellular reactive oxygen species concentration, by an electron transfer-based colorimetric cupric-reducing antioxidant capacity assay justifying that cellular total antioxidant capacity which uses all the combined antioxidant activities present within vitamins, proteins, lipids, and glutathione reverses these deleterious stress effects. Henceforth, performance of Z. bisporus MTCC 4801 mounted because of stress regime seems to be multifactorial.

The role of H+/OH− channels in saline pathology of Chara australis: brief history

The pH banding pattern, observed in Chara and Nitella early last century, suggests that there are H+ or OH− conducting channels in the alkaline bands of the characean plasma membrane. If the cells are exposed to high pH medium, pHo, these channels become the dominant membrane conductance: High pH state. Goldman-Hodgkin-Katz (GHK) equation can model the current-voltage (I/V) characteristics of this state with either H+ or OH− as permeant ion. However, H+ requires unrealistically large increases in channel conductance/number parameter as pHo increases. Recently we discovered a typical spiky noise in the membrane potential difference (PD) of salt sensitive Chara australis, appearing immediately upon exposure to saline. The H+/OH− channels are thought to open transiently in small patches all over the cell, while negative membrane PD does not support their open state/s. As the membrane PD depolarizes due to inhibition of the proton pump by salinity, the H+/OH− channels remain open to dominate the I/V characteristics, which can also be modeled by GHK equation. The addition of 1 mM ZnCl2, the main known blocker of animal H+ channels, abolishes both the PD noise and the H+/OH− I/V characteristics in high pH state and in salt stressed cells. Confocal microscopy and fluorescein isothiocyanate (FITC)-dextran illuminate the pH changes around cells and reveal transient bright spots early in saline exposure, becoming more numerous and fixed in space with time in saline APW.

Metabolic response of Tetragenococcus halophilus under salt stress

In this study, the effect of salt stress on metabolic response of Tetragenecoccus halophilus was investigated, and the metabolic alternations were analyzed using liquid chromatography-mass spectrometry according to the metabolomics approach. A total of 81 intracellular metabolites were identified, and significant differences were observed in the levels of metabolites mainly participating in central carbon metabolism, fatty acid metabolism, and amino acid metabolism. Analysis of the membrane fatty acid distribution showed that higher proportions of unsaturated fatty acid were observed in salt-treated cells. Additionally, salt-stressed cells exhibited higher amounts of compatible solutes including proline, glycine, citrulline, and N-acetyltyrptophan, and lower amounts of branched-chain amino acids. Interestingly, higher amounts of indole, salicylic acid, and coronatine, which are regarded as signaling molecule and suggested to combat osmotic stress, were detected in salt-shocked cells compared with the untreated cells. Taken together, these results suggested that increased unsaturated membrane fatty acids, accumulation of compatible solutes, and up-regulation of signaling molecule may be potential mechanisms employed by T. halophilus during salt stress.

Physiological and anatomical responses of Phyllostachys vivax and Arundinaria fortunei (Gramineae) under salt stress

This research was conducted in order to study salt tolerance of Phyllostachys vivax McClure and Arundinaria fortunei (Van Houtte) Riviere et C. Riviere. The effects of salinity stress on growth parameters, anatomical changes, and ion accumulation from two 1-year-old bamboo species under different salt treatments were investigated. The growths of P. vivax and A. fortunei were not severely inhibited when salt content was lower than 0.42%. Their structural properties of vascular tissue were affected, and both species showed decreases in vessel diameters and a higher vessel density under salt stress, but they could still maintain relatively stable anatomical structure in saline soils. Besides, the results indicated that Na+ accumulation progressively increased for organs as salinity levels increased. In particular, the accumulation of Na+ in underground organs was obviously higher than those in culms and leaves. The levels of K+, Ca2+, and Mg2+ in salt-stressed cells gradually decreased in response to the increase of Na+. Overall, we concluded that P. vivax and A. fortunei had the ability to withstand salt stress.

Extracellular ATP mediates cellular K+/Na+ homeostasis in two contrasting poplar species under NaCl stress

eATP mediates cellular K + and Na + homeostasis in two contrasting poplar species differing in salt tolerance. Using the non-invasive micro-test technology (NMT), the effects of extracellular ATP (eATP) on salt-altered flux profiles of K+, Na+, and H+ were investigated in salt-tolerant poplar species, Populus euphratica and salt-sensitive P. popularis. A short-term NaCl (100 mM NaCl, 12 h) resulted in a Na+ efflux and a correspondingly increased H+ influx in P. euphratica cells, but the effect was not seen in P. popularis. ATP (50 μM) enhanced exchange of Na+ with H+ in salt-stressed cells of two species (6, 12 h), especially in P. popularis. However, the ATP-stimulated Na+ efflux and H+ influx were significantly inhibited by amiloride (a Na+/H+ antiporter inhibitor) or sodium orthovanadate (a plasma membrane H+-ATPase inhibitor), indicating that the ATP induction of Na+ extrusion resulted from an active Na+/H+ antiport across the plasma membrane (PM). NaCl accelerated K+ efflux in the two species, with a more pronounced effect in the salt-sensitive poplar. The salt-induced K+ efflux was markedly restricted by the K+ channel blocker, tetraethylammonium chloride, indicating that the K+ efflux is mediated by depolarization-activated outward rectifying K+ channels and non-selective cation channels. ATP benifited poplar cells, especially the salt-sensitive P. popularis, in maintaining K+ homeostasis under external salinity. This was likely the result of activated H+ pump in the PM, which restricted the K+ efflux through the inhibition of depolarization-activated K+ channels in both species. Na+ and K+ flux recordings revealed that non-hydrolysing analogues of ATP, αβ-meATP (50 μM), and ATPγS (50 μM) produced an effect similar to that of the hydrolysable form but with a more pronounced effect. However, ADP- and AMP-stimulated cells (50 μM) exhibited behaviors different from those invoked by ATP, αβ-meATP, and ATPγS treatments. eATP signalling in K+ and Na+ homeostasis was blocked by the antagonists of animal P2 receptors, PPADS, and suramin. Moreover, ATP-stimulated Na+ extrusion and reduction of K+ loss in NaCl-stressed cells were inhibited by LaCl3 (an inhibitor of Ca2+-permeable channels) and DPI (an inhibitor of PM NADPH oxidase), indicating that ATP signalling was mediated via second messengers, H2O2 and Ca2+, in the two poplars differing in salt tolerance.

Photosynthetic and extracellular production of glucosylglycerol by genetically engineered and gel-encapsulated cyanobacteria.

Glucosylglycerol (GG) has a range of potential applications in health, pharmacy, and cosmetics due to its physiological, protein-stabilizing, and antioxidative properties. In addition to chemical synthesis and enzymatic catalysis, GG can be produced as a protective osmolyte in salt-stressed bacteria, such as the cyanobacterium Synechocystis sp. PCC 6803. Here, we presented an efficient GG production and secretion by genetically modified and encapsulated Synechocystis cells grown in a semicontinuous manner. We improved the production and secretion of GG in Synechocystis by first disrupting both the ggtC and ggtD genes, which encode the subunits of a GG uptake transporter, as well as the ggpR gene, which encodes a repressor for GG synthesis. Then, we confirmed that the rapid GG release from salt-stressed cells of Synechocystis depended on the ion gradient across the cell membrane. Finally, we proved the feasibility of an agar gel encapsulation method in supporting cell growth and the GG production of Synechocystis under semicontinuous culturing conditions.

Microarray and suppression subtractive hybridization analyses of gene expression in hybrid poplar (Populus alba × Populus tremula var. glandulosa) cell suspension cultures after exposure to NaCl

The gene expression profiles of hybrid poplar (Populus alba×Populus tremula var. glandulosa) cells in suspension culture after exposure to salinity (NaCl) induced stress were examined by constructing two suppression subtractive hybridization (SSH) libraries. cDNA from non-treated cells was used as a driver and cDNA samples from cell suspension cultures exposed to 150mM NaCl for 2 or 10h were used as testers. Randomly selected clones from each SSH library were sequenced and 727 high-quality expressed sequence tags (ESTs) were obtained and analyzed. Four novel ESTs were identified. Between the two libraries, 542 unique SSH clones were selected for placement on a cDNA microarray. In total, 18 differentially expressed genes were identified with 4 and 12 genes being significantly differentially expressed 2 and 10h after the treatment, respectively. Genes related to metabolism and protein synthesis and several genes whose protein products are implicated in salt or other abiotic stress-related responses were expressed in the salt-stressed cells.

Volatile communication between Chlamydomonas reinhardtii cells under salt stress

Although it is known that volatile organic compounds (VOCs) can be released by lower plants, their release and ecological functions are not much studied, especially in aquatic systems under abiotic stress. In the present study, we determined the compositions of VOCs released from Chlamydomonas reinhardtii under both neutral (NaCl) and alkali (Na2CO3) salt stresses. Hexanal was the newly released abundant compound under NaCl stress, while we found 3,4-dimethyl-hexane and 5-methyl-2-heptene under Na2CO3 stress. Under both stress conditions, longifolene was the significantly increased compound. When C. reinhardtii cells were exposed to the VOCs from the salt-stressed cells, the cell density was reduced, but the content of chlorophyll, Fv/Fm and antioxidant enzyme activity were increased. This might indicate that those VOCs can transfer stress signal between C. reinhardtii cells and tell the receptor to prepare against reactive oxygen species (ROS) damage ahead of the stress coming.