Long-term hyposaline and hypersaline stresses produce distinct antioxidant responses in the marine alga Dunaliella tertiolecta

Tolerance to salinity stress in higher plants correlates to levels of antioxidant enzymes and/or substrates. Do hyperosmotic and hypoosmotic stress induce antioxidant responses in salt tolerant algae, and if so, are these responses the same for both excess and minimal salinity? To answer these questions, cultures of the marine alga Dunaliella tertiolecta (Chlorophyta) were grown in seven salinities covering a 60-fold range from 0.05 to 3.0 mol/L NaCl. Long-term effects of salinity on growth and antioxidant parameters were determined. Growth rates were reduced at the salinity extremes (0.05 mol/L NaCl and 3 mol/L NaCl) indicating the cultures were stressed. The levels of six antioxidant enzymes and three antioxidant substrates were quantified at these growth salinities. Compared to growth at optimum salinities (i.e. 0.2-0.5 mol/L NaCl), high salinities produced a 260% increase in monodehydroascorbate reductase, a doubling of ascorbate peroxidase activity and a three-fold increase in the rate of dark respiration. Cells acclimated to low growth salinities (hyposaline stress, i.e. < 0.2 mol/L NaCl) showed major increases in glutathione and alpha-tocopherol coupled with decreases in Fv/Fm ratios and in total and reduced ascorbate compared to moderate and high external salinities. Cell volumes remained unchanged, except at the lowest salinity where they doubled. Catalase, superoxide dismutase, dehydroascorbate reductase and glutathione reductase activities were not altered by extreme salinities. The involvement of oxidative stress at both salinity extremes is implied by the alterations in antioxidant enzymes and substrates, but the specific changes are very different between hypo and hypersaline stresses.     

Identification and characterization of a new strain of the unicellular green alga Dunaliella salina (Teod.) from Korea

The unicellular green alga Dunaliella salina is a halotolerant eukaryotic organism. Its halophytic properties provide an important advantage for open pond mass cultivation, since D. salina can be grown selectively. D. salina was originally described by E. C. Teodoresco in 1905. Since that time, numerous isolates of D. salina have been identified from hypersaline environments on different continents. The new Dunaliella strain used for this study was isolated from the salt farm area of the west coastal side of South Korea. Cells of the new strain were approximately oval- or pear-shaped (approximately 16-24 microm long and 10-15 microm wide), and contained one pyrenoid, cytoplasmatic granules, and no visible eyespot. Although levels of beta-carotene per cell were relatively low in cells grown at salinities between 0.5 to 2.5 M NaCl, cells grown at 4.5 M NaCl contained about a ten-fold increase in cellular levels of beta-carotene, which demonstrated that cells of the new Korean strain of Dunaliella can overaccumulate beta- carotene in response to salt stress. Analysis of the ITS1 and ITS2 regions of the new Korean isolate showed that it is in the same clade as D. salina. Consequently, based on comparative cell morphology, biochemistry, and molecular phylogeny, the new Dunaliella isolate from South Korea was classified as D. salina KCTC10654BP.     

不同盐度胁迫下杜氏盐藻全转录组测序及注释

【目的】通过对杜氏盐藻的转录组进行测序和基因功能分析,阐明不同浓度盐胁迫对杜氏盐藻生长发育以及不同信号途径的影响。【方法】分别获取9%NaCl浓度和24%NaCl浓度培养下的杜氏盐藻转录组并通过Illumina平台进行测序。将所得的序列进行拼接、去冗余处理。【结果】获得40682个unigenes,其中注释到NR数据库的10905个,注释到NT数据库的2768个,注释到SWISS-PROT数据库的7261个,注释到COG/KOG数据库的6499个。受到高盐胁迫的杜氏盐藻细胞相比低盐环境下,有717个基因表达上调,1012个基因表达下调。进一步对60个显著差异基因进行了功能聚类,发现盐胁迫诱导了光合作用途径的基因表达。【结论】杜氏盐藻通过提高光合作用基因表达增强耐盐性。该研究最大范围上挖掘了杜氏盐藻在高盐和低盐环境的基因转录水平,为深入揭示杜氏盐藻盐胁迫下基因差异表达提供了平台,并为进一步研究杜氏盐藻耐盐机理提供理论依据。     

A Salt-Induced 60-Kilodalton Plasma Membrane Protein Plays a Potential Role in the Extreme Halotolerance of the Alga Dunaliella

The halotolerant alga Dunaliella salina grows in saline conditions as varied as 0.5 and 5 M NaCl, maintaining throughout this range a low intracellular ion concentration. To discover factors potentially involved in ionic homeostasis, we grew cells in media with different salinities or osmolarities and compared their protein profiles. The comparisons indicated that the amount of a 60-kD protein, p60, greatly increased with an increase in salinity and was moderately enhanced when NaCl was substituted with iso-osmotic glycerol. Cells transferred from low to high NaCl or from high glycerol to iso-osmotic NaCl media transiently ceased to grow, and resumption of growth coincided approximately with an increase in p60. The protein, extracted from a plasma membrane fraction, was purified to homogeneity. Anti-p60 antibodies cross-reacted with a 60-kD protein in Dunaliella bardawil. Immunoelectron microscopy of D. salina cell sections indicated that p60 was exclusively located in the plasma membrane. Its induction by salt, the correlation between its accumulation and growth resumption in high concentrations of salt, and its plasma membrane localization suggest the possibility that p60 could play a role in ionic homeostasis in conditions of high salinity, although different types of function could also be considered.     

A novel limb in the osmoregulatory network of Methanosarcina mazei Gö1: Nε-acetyl-β-lysine can be substituted by glutamate and alanine

N^ε-acetyl-β-lysine is a unique compatible solute found in methanogenic archaea grown at high salinities. Deletion of the genes that encode the lysine-2,3-aminomutase ( ablA ) and the β-lysine acetyltransferase ( ablB ) abolished the production of N^ε-acetyl-β-lysine in Methanosarcina mazei Gö1. The mutant grew well at low and intermediate salinities. Interestingly, growth at high salt (800 mM NaCl) was only slowed down but not impaired demonstrating that in M. mazei Gö1 N^ε-acetyl-β-lysine is not essential for growth at high salinities. Nuclear magnetic resonance (NMR) analysis revealed an increased glutamate pool in the mutant. In addition to α-glutamate, a novel solute, alanine, was produced. The intracellular alanine concentration was as high as 0.36 ± 0.05 μmol (mg protein)⁻¹ representing up to 18% of the total solute pool at 800 mM NaCl. The cellular alanine concentration increased with the salinity of the medium and decreased in the presence of glycine betaine in the medium, indicating that alanine is used as compatible solute by M. mazei Gö1.     

SALINITY ADAPTATION BY DUNALIELLA TERTIOLECTA. I. INCREASES IN CARBONIC ANHYDRASE ACTIVITY AND EVIDENCE FOR A LIGHT-DEPENDENT Na+/H+ EXCHANGE1,2

When Dunaliella tertiolecta, previously adapted to medium containing 0.5 M NaCl, is transferred to higher salinities, there is a lag in growth, suggesting an adaptation period. Since there is no significant difference in the Na⁺ content of cells grown between 0.5 and 3.5 M NaCl, a mechanism for Na⁺ extrusion or exclusion is indicated. Increasing the salinity of cell suspensions stimulates an incorporation of H⁺ by the cells, suggesting an H⁺/Na⁺ exchange. Cells adapted to higher salinities have, increased carbonic anhydrase activity, suggesting that increased CO₂ or HCO₃^? transport may be required at higher salinities. Growth, of D. tertiolecta at salinities above 2.5 M requires continuous illumination; therefore a light-driven H⁺/Na⁺ exchange accompanied by a HCO₃^? influx is proposed.     

Salt induction of fatty acid elongase and membrane lipid modifications in the extreme halotolerant alga Dunaliella salina

In studies of the outstanding salt tolerance of the unicellular green alga Dunaliella salina, we isolated a cDNA for a salt-inducible mRNA encoding a protein homologous to plant beta-ketoacyl-coenzyme A (CoA) synthases (Kcs). These microsomal enzymes catalyze the condensation of malonyl-CoA with acyl-CoA, the first and rate-limiting step in fatty acid elongation. Kcs activity, localized to a D. salina microsomal fraction, increased in cells transferred from 0.5 to 3.5 M NaCl, as did the level of the kcs mRNA. The function of the kcs gene product was directly demonstrated by the condensing activity exhibited by Escherichia coli cells expressing the kcs cDNA. The effect of salinity on kcs expression in D. salina suggested the possibility that salt adaptation entailed modifications in the fatty acid composition of algal membranes. Lipid analyses indicated that microsomes, but not plasma membranes or thylakoids, from cells grown in 3.5 M NaCl contained a considerably higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids compared with cells grown in 0.5 M salt. Thus, the salt-inducible Kcs, jointly with fatty acid desaturases, may play a role in adapting intracellular membrane compartments to function in the high internal glycerol concentrations balancing the external osmotic pressure.     

Relationship between the photosynthetic activity and the performance of Cakile maritima after long-term salt treatment

Cakile maritima is a halophyte with potential for ecological, economical and medicinal uses. We address here the impact of salinity on its growth, photosynthesis and seed quality. Whole plant growth rate and shoot development were stimulated at moderate salinity (100–200 m M NaCl) and inhibited at higher salt concentrations. Although diminished in the presence of salt, potassium and calcium uptake per unit of root biomass was maintained at relatively high value, while nutrient-use efficiency (NUE) was improved in salt-treated plants. Chl and carotenoid concentrations decreased at extreme salinities, but anthocyanin concentration continuously grew with salinity. Net photosynthetic rate (A), stomatal conductance, maximum quantum efficiency of PSII and quantum yield were stimulated in the 100–200 m M NaCl range. Higher salinity adversely affected gas exchange and changed PSII functional characteristics, resulting in a reduction of A per leaf area unit. This phenomenon was associated with increased non-photochemical quenching. Harvest index, silique number and seeds per fruit valve were maximal at 100 m M NaCl. Despite the decreasing salt accumulation gradient from the vegetative to the reproductive organs, high salinities were detrimental for the seed viability and increased the proportion of empty siliques. Overall, the salt-induced changes in the plant photosynthetic activity resulted into analogous responses at the vegetative and reproductive stages. The enhancement of NUE, the absence of pigment degradation, the reduction of water loss and the concomitant PSII protection from photodamage through thermal dissipation of excess excitation significantly accounted for Cakile survival capacity at high salinity.     

Growth phase-dependent switch in osmolyte strategy in a moderate halophile: ectoine is a minor osmolyte but major stationary phase solute in Halobacillus halophilus

The moderately halophilic, chloride-dependent bacterium Halobacillus halophilus switches its osmolyte strategy with the salinity in its environment by the production of different compatible solutes. Ectoine is produced predominantly at very high salinities, along with proline. Interestingly, ectoine production is growth phase dependent which led to a more than 1000-fold change in the ectoine : proline ratio from 0.04 in exponential to 27.4 in late stationary phase cultures. The genes encoding the ectoine biosynthesis pathway were identified on the chromosome in the order ectABC . They form an operon that is expressed in a salinity-dependent manner with low-level expression below 1.5 M NaCl but 10-fold and 23-fold increased expression at 2.5 and 3.0 M NaCl respectively. The temporal expression of genes involved in osmoresponse is different with gdh / gln and pro genes being first, followed by ect genes. Chloride had no effect on expression of ect genes, but stimulated cellular EctC synthesis as well as ectoine production. These data demonstrate, for the first time, a growth-phase dependent switch in osmolyte strategy in a moderate halophile and, additionally, represent another piece of the chloride regulon of H. halophilus .     

OPTIMIZATION OF SALT CONCENTRATIONS FOR A HIGHER CAROTENOID PRODUCTION IN DUNALIELLA SALINA (CHLOROPHYCEAE)1

Dunaliella salina (Dunal) Teodor, when treated over 25 d with a wide range of NaCl salinities (0.6–4.5 M), showed its maximal growth potentialities at 1.5–3.0 M NaCl and was able to survive even at 4.5 M NaCl. Sodium concentrations increased significantly at the supraoptimal salinities, reaching up to 5 mmol · g^?1 dry weight (dwt) at 4.5 M NaCl. Interestingly, ability of D. salina to take up essential mineral nutrients was not impaired by increased salinity. As for growth, chl concentrations were maximal in the 1.5–3.0 M NaCl range. Interestingly, carotenoid concentrations increased with the increasing salinity. The highest values of total antioxidant activity (5.2–6.9 mg gallic acid equivalents [GAE] · g^?1 dwt), antiradical activity, and reducing power were measured at 1.5–3.0 M NaCl. As a whole, these results showed that at 1.5–3.0 M NaCl, D. salina produce appreciable antioxidant level. But, once it reaches its growth maximum, a salt addition up to 4.5 M could enhance its carotenoid yield.     

Biochemical and physiological characterization of a halotolerant Dunaliella salina isolated from hypersaline Sambhar Lake,India

The objective of the present study was to characterize intrinsic physiological and biochemical properties of the wall‐less unicellular cholorophyte Dunaliella salina isolated from a hypersaline Sambhar Lake. The strain grew optimally at 0.5 M NaCl and 16:8 h L:D photoperiod along with maintaining low level of intracellular Na⁺ even at higher salinity, emphasizing special features of its cell membranes. It was observed that the cells experienced stress beyond 2 M NaCl as evidenced by increased intracellular reactive oxygen species and antioxidative enzymes, nevertheless proline and malondialdehyde content declined sharply accompanied by higher neutral lipid accumulation. Salinity exceeding 2 M resulted decrease in photosynthetic quantum yield (Fv/Fm) and enhanced glycerol synthesis accompanied by leakage. Super oxide dismutase seemed to play a pivotal role in antioxidative defense as eight isoforms were expressed differentially while catalase and glutathione peroxidase showing no significant change in their expression at higher salinity. The ability of D. salina to grow in range of salinities by sustaining healthy photosynthetic apparatus along with accumulation of valuable products made this alga an ideal organism that can be exploited as resource for biofuel and commercial products.     

Effect of nitrate concentration on growth and pigment synthesis of Dunaliella salina cultivated under low illumination and preadapted to different salinities

A wild strain of Dunaliella salina was isolated from a solar evaporation salt-pond in Araya (Estado Sucre, Venezuela) and grown in batch culture using relatively low illumination (80 μmol photon m-2 s-1). After the alga had been adapted to various salinities (9, 14, 21% w/v NaCl), the influence of nitrate concentration (882, 435, 212 μmol L-1 N) on growth rate and chlorophyll a and total carotenoid concentrations was measured. Low nitrate concentration negatively affected growth, but enhanced carotenoid accumulation. A slight increase in carotenogenesis was also observed in alga grown at the highest salinity. There were no significant additive or synergistic effects of salinity and nutrient concentrations on the concentrations of chlorophyll a or total carotenoid. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modification of the salinity response of wheat by the genome ofElytrigia elongatum

Summary Triticum aestivum cv. Chinese Spring wheat,Elytrigia elongatum (tall wheatgrass), and theTriticum-Elytrigia amphiploid were grown in complete nutrient culture containing, in addition, 0, 40, 80 and 120 mM NaCl. The 3 genotypes responded quite differently to increasing salinity; the Na concentration of wheat shoots increased in direct proportion to the increase in salinity of the external medium whereas the Elytrigia response was interpreted as showing high affinity for Na at low external Na (40 mM) but comparative exclusion of Na at high salinities (120 mM). In contrast, Na levels of the amphiploid were less than those of either wheat or Elytrigia under both low and high salinities. Thus the amphiploid behaved like wheat at 40 mM NaCl but more like Elytrigia at 120 mM NaCl because Na transport to the amphiploid shoot was restricted over the whole salinity range. The K concentration of the amphiploid shoot at high salinities was significantly greater than the K concentrations of either wheat or Elytrigia.     

Effects of salinity on beta-carotene production by Dunaliella tertiolecta DCCBC26 isolated from the Urmia salt lake, north of Iran

This study examined the effect of different salt concentrations (0.05-3 M of NaCl) on the kinetics of growth, total carotenoids and beta-carotene (all-trans and 9-cis) accumulated in Dunaliella tertiolecta DCCBC26, a microalgae strain isolated from the Urmia hypersaline lake, northwest of Iran. Results indicated that the highest amount of carotenoids detected (11.73 mg/l) was in the salinity of 0.5 M NaCl during the stationary growth phase. The percentage of the all-trans and 9-cis-beta-carotene in the exponential phase were 92% and 32% in salinities of 3 M and 0.5 M, respectively. However, only 23% of the beta-carotene was detected in the stationary growth phase of the microalgae in 0.5 M salinity and was 9-cis isomer.     

Variation in gene expression along a salinity gradient in wild populations of the euryhaline black-chinned tilapia Sarotherodon melanotheron

This study evaluated variation in expression of 11 genes within and among six wild populations of the black-chinned tilapia Sarotherodon melanotheron distributed along a salinity gradient from 0 to 100. Previous laboratory studies had shown that expression of these genes was sensitive to water salinity; the current study confirmed that a number of them also varied in expression in wild populations along the salinity gradient. Principal component analysis (PCA) first distinguished two, not mutually exclusive, sets of genes: trade-off genes that were highly expressed at one or other extreme of the salinity gradient and stress genes that were up-regulated at the two salinity extremes (i.e. a U-shaped expression pattern). The PCA clearly partitioned the populations into three groups based on their gene expression patterns and their position along the salinity gradient: a freshwater (GL; 0) population, four brackish and seawater (GB, HB, SM, SF; ranging from 20 to 50) populations and a hypersaline (SK, 100) population. Individual variation in gene expression was significantly greater within the populations at the extreme compared to intermediate salinities. These results reveal phenotypically plastic regulation of gene expression in S. melanotheron, and greater osmoregulatory and plasticity costs at extreme salinities, where fitness-related traits are known to be altered.     

ESR studies on the membrane properties of a moderately halophilic bacterium. II. Effect of extreme growth conditions on liposome properties

Lipid preparations from the cells of a moderately halophilic bacterium, Pseudomonas halosaccharolytica grown under the two extreme conditions of high temperature-high NaCl concentration and low temperature-low NaCl concentration showed distinctively different profiles in phospholipid and fatty acid composition. Cells grown at 40 degrees C in medium containing 3.5 M NaCl had high concentrations of saturated and C19 cyclopropanoic fatty acids (about 50 per cent of the total), whereas cells grown at 20 degrees C in medium containing 0.5 M NaCl had decreased concentrations of these fatty acids with increased concentrations of the corresponding unsaturated fatty acids. The phospholipid composition was also affected ty the culture conditions; cells grown at 40 degrees C in 3.5 M NaCl had large amounts of acidic phospholipids, whereas those grown at 20 degrees C in 0.5 M NaCl had small amounts. ESR studies on liposomes prepared from lipids of cells grown under the two conditions showed characteristic profiles for correlation times and order parameters of three spin labels of stearic acid derivatives similar to those of membranes of whole cells of this bacterium. ESR studies showed that the physical properties of the liposomes from the total extractable lipids and isolated phosphatidylglycerol from the cells were completely different from those of synthetic dioleoylphosphatidylglycerol. Liposomes of the lipids extracted from cells grown at 40 degrees C in 3.5 M NaCl showed change in rotational viscosity on altering the NaCl concentration to 0.5M, whereas liposomes of lipids extracted from cells grown at 20 degrees C in 0.5 M NaCl did not show change in rotational viscosity on increasing the NaCl concentration to 3.5 M.