The Role of Glycerol in the Osmotic Regulation of the Halophilic Alga Dunaliella parva

Dunaliella parva, a green halophilic alga, was found to accumulate very large amounts of intracellular glycerol. Through measurements of the intracellular volume the internal concentration of glycerol was calculated and found to be around 2.1 m in cells cultured in 1.5 m NaCl. When the extracellular salt concentration of an algal suspension was increased or decreased, the intracellular glycerol varied accordingly, reaching its new osmotic equilibrium after about 90 minutes. Since no leakage of intracellular glycerol was observed above 0.6 m NaCl, these alterations in glycerol content are interpreted as due to metabolic formation and degradation of intracellular glycerol. The above results indicate the existence of a new type of algal osmoregulation, in which the osmotic balance depends on the synthesis or degradation of intracellular glycerol in response to the external salt concentration.     

Glycerol fomation inDunaliella cells under non-growing conditions with hypertonic lithium or magnesium media

Glycerol formation ofDunaliella cells in non-growing media was investigated.Dunaliella tertiolecta andD. bioculata grew well in a NaCl medium but not at all in a LiCl or a MgCl₂ medium. When the cells originally suspended in a medium containing 0.5 M NaCl were transferred to media which contained one of 1 M NaCl, 1 M LiCl or 0.7 M MgCl₂, the intracellular glycerol content increased.D. tertiolecta cultured in either a 1 M LiCl or a 0.7 M MgCl₂ medium did not multiply, but maintained abilities to evolve O₂ in the light and absorb O₂ in thedark even after about a 5 day culture. From these results, it can be concluded that the halotolerance ofDunaliella to different kinds of salts is not directly related to osmoregulation by the glycerol formation.     

The size and turnover of the glycerol pool in Dunaliella

Abstract The rate of incorporation of ¹⁴C from CO₂ into glycerol, and the amount of glycerol in cells of Dunaliella tertiolecta was determined at constant salinity at two representative concentrations of NaCl. From these data, the rate of synthesis and turnover of the glycerol pool was determined. The half-time for turnover of the glycerol pool was of the order of 1 h in 170 mol m^?3 NaCl and almost 6h for 700 mol m^?3 NaCl. These results indicate that turnover of the glycerol pool in Dunaliella is relatively slow under steady-state conditions. Synthesis and dissimilation of glycerol do not apparently constitute a metabolic cycle in the conventional sense. Rather, glycerol metabolism resembles that of the storage polysaccharides which arc commonly produced and degraded by different pathways.     

THE GLYCEROL PERMEABILITY OF THE PLASMALEMMA OF THE HALOTOLERANT GREEN ALGA DUNALIELLA PARVA (VOLVOCALES)1

The glycerol permeability of the plasmalemma of the green alga Dunaliella parva Lerche was investigated by efflux studies with labelled glycerol, by enzymatic determination of glycerol leakage, and the determination of the reflection coefficient from osmotically induced volume changes (zero flow method). All results indicate that the plasmalemma of D. parva does not exhibit a special low permeability towards glycerol as would be expected from a glycerol accumulating alga. Rather, significant amounts of glycerol diffuse continuously into the medium following the glycerol concentration gradient between the cells and the medium. Efflux rates vary between 0.1 and 2 μmoles glycerol·mg^?1 chlorophyll·h^?1 depending on the external NaCl concentration. After one day up to 25% of the total glycerol of the algal suspension was found in the medium. Within 10 days this value can increase to 60%, depending on the growth constant of the culture. The reflection coefficient σ was determined to be 0.87, the permeability coefficient 2800 × 10^?11 m·sec^?1. To maintain a proper endogenous glycerol level corresponding to the external osmotic pressure, glycerol efflux in D. parva has to be balanced by a continuous synthesis of glycerol. D. parva follows the strategy of “glycerol efflux tolerance” instead of “glycerol efflux avoidance”. The alga has to pay the energetic costs of this strategy of tolerance.     

Responses of photosynthetic apparatus of the halotolerant microalga <Emphasis Type="Italic">Dunalliella maritima</Emphasis> to hyperosmotic salt shock

Chlorophyll fluorescence induction curves were used as a means to assess the functional condition of the photosynthetic apparatus in cells of the halotolerant green microalga Dunaliella maritima (Massjuk) (division Chlorophyta) exposed to hyperosmotic salt shock of various intensities. The shock was caused by the transfer of algal cells grown in the medium with 0.5 M NaCl to the media with elevated NaCl concentrations (1.0, 1.5, and 2.0 M). Parameters of chlorophyll fluorescence (F ₀, F _m, F ₀′, F _t′) were measured by means of a specialized pulse-amplitude-modulation fluorometer PAM 2100. In addition, the rate of photosynthetic oxygen evolution as well as the intracellular Na⁺ and glycerol content (the main osmolyte in this microalga) were determined. The hyperosmotic salt shock was found to elevate the intracellular Na+ content and reduce the functional activity of PSII in D. maritima. The suppression of PSII activity was evident from the decrease in the maximal quantum yield of photochemical energy conversion in PSII, the decreased rate of linear electron transport, the increased reduction of the primary acceptor Q_A, and the suppression of photosynthetic O₂ evolution. The functional activity of PSII recovered gradually along with restoration of osmotic and ionic balance in algal cells. It is proposed that PSI ensures energy supply during cell responses of D. maritima to hyperosmotic salt shock.     

Effect of temperature on glycerol retention in the halotolerant algae dunaliella and asteromonas

Algae of the genera Dunaliella and Asteromonas can maintain extremely high concentration gradients (>10⁴) of glycerol between the intracellular space and the medium. This unique ability is highly temperature-dependent. Treating the algae for several minutes at temperatures exceeding 60 C causes complete release of all the internally held glycerol; 50% release occurs around 50 C, but essentially none is released below 40 C. A similar behavior was observed in several species of Dunaliella, and one of Asteromonas and is independent of the salt concentration of the medium. The underlying mechanism may involve a temperature-dependent conformational transition of a component of the cellular membrane which is essential for glycerol impermeability.     

Osmoregulation in the Halotolerant Alga Asteromonas gracilis

Asteromonas gracilis, a green wall-less halotolerant alga, grows on salt concentrations from 0.5 molar NaCl (seawater) to saturation (4.5 molar NaCl). The specific growth rate was maximal at concentrations between 0.5 and 2.5 molar and only gradually decreased above 2.5 molar. Photosynthetic oxygen evolution was maximal over a range of salinities around 2.5 molar and the photosynthesis to respiration ratio showed a maximum at 1.5 molar NaCl. The alga accumulates large amounts of intracellular glycerol in response to saline conditions. The glycerol content of the cells varied in direct proportion to the extracellular salt concentration, being about 50 and 400 picograms glycerol per cell in algae grown at 0.5 and 4.5 molar NaCl, respectively. In salt concentrations lower than 3.5 molar and at growth temperatures below 40 C, essentially all the glycerol was intracellular. Above 3.5 molar NaCl, about 25 per cent of the total glycerol leaked slowly from the cells to the medium. Treating the algae for several minutes at temperatures exceeding 47 C caused 50 per cent release of the internal glycerol. At 60 C, 100 per cent of the glycerol was released. When the extracellular salt concentration was increased or decreased, the intracellular glycerol varied accordingly, reaching its new intracellular level after a few hours. Both photosynthesis and respiration were inhibited on transfer of the cells from 1.5 to 3.5 molar NaCl but were not inhibited on transfer of the cells from 3.5 to 1.5 molar NaCl. The kinetics of photosynthetic resumption preceded the kinetics of glycerol biosynthesis. The above results indicate the existence of osmotic regulations in Asteromonas gracilis via the accumulation of intracellular glycerol.     

Expression of the Chlamydomonas reinhardtii Sedoheptulose‐1,7‐bisphosphatase in Dunaliella bardawil leads to enhanced photosynthesis and increased glycerol production

Bioengineering of photoautotrophic microalgae into CO₂ scrubbers and producers of value‐added metabolites is an appealing approach in low‐carbon economy. A strategy for microalgal bioengineering is to enhance the photosynthetic carbon assimilation through genetically modifying the photosynthetic pathways. The halotolerant microalgae Dunaliella posses an unique osmoregulatory mechanism, which accumulates intracellular glycerol in response to extracellular hyperosmotic stresses. In our study, the Calvin cycle enzyme sedoheptulose 1,7‐bisphosphatase from Chlamydomonas reinhardtii (CrSBPase) was transformed into Dunaliella bardawil, and the transformant CrSBP showed improved photosynthetic performance along with increased total organic carbon content and the osmoticum glycerol production. The results demonstrate that the potential of photosynthetic microalgae as CO₂ removers could be enhanced through modifying the photosynthetic carbon reduction cycle, with glycerol as the carbon sink.     

Effects of NaCl and KNO3 concentrations on the abscisic acid content of Dunaliella sp. (Chlorophyta)

Abscisic acid (ABA) is a hormone which has a number of roles during the life cycle of a plant. We demonstrated the occurrence of ABA in a halotolerant green alga, Dunaliella sp. isolated from a salt pond near Adelaide, South Australia, using thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The variation of cellular ABA and protein content during the growth of an axenic clonal culture of Dunaliella sp. was investigated under different concentrations of NaCl and KNO₃.Experimental results can be summarized as follow: (1) ABA content was changed with the growth stage of culture: A rapid increase in ABA content was observed in the logarithmic phase. After this, the content rapidly decreased to very low values. (2) ABA content was also affected by the NaCl concentration. The content had a minimum value at the NaCl concentration (15%) where growth rate was maximal, and higher values at higher or lower concentrations of NaCl. (3) The ABA content also increased with decreasing nitrogen concentration of the medium.     

Endopolyploidy in Chlorella

A mutant of Dunaliella tertiolecta produced by treatment with methyl nitrosoguanidine and designated HL25/8, grew more slowly than the parent strain under all experimental conditions and was conspicuously less tolerant of NaCl. Total photosynthetic activity (C-fixation and O₂ evolution) was less in HL25/8 than in the parent strain and was affected differently by [NaCl] in the two strains. Various growth characteristics indicated that the mutant had a greater need than the parent strain for CO₂ as distinct from HCO ₃ ^– as a source of carbon. Gaseous CO₂ extended the range of salt tolerance of the mutant. For example, HL25/8 could not sustain growth at 1.02 M NaCl in a conventional buffered medium containing bicarbonate as the sole carbon source but could do so if the medium were sparged with a CO₂/air mixture. The mutant strain has a lower activity of carbonic anhydrase on the cell surface than the parent D. tertiolecta. Moreover, the two strains differ sharply in the responses of their surface carbonic anhydrase activity to salinity of the growth medium. Increasing sodium chloride concentration above 0.17 M raised activity of the enzyme in the parent strain but decreased it in HL25/8. We conclude that the low activity of carbonic anhydrase and its response to salinity can largely, but perhaps not fully, explain the diminished salt tolerance of the mutant. A plate counting method applicable to Dunaliella is described.     

Photosynthetic Activities of the Halophilic Alga Dunaliella parva

Dunaliella parva, a unicellular halophilic alga, was found to evolve oxygen photosynthetically only in the presence of a high osmolar concentration. Cell free preparations were obtained by placing the cells in a medium of low osmolarity. The fragments obtained showed a high photoreducing and photophosphorylating activity except for their inability to catalyze all ferredoxin dependent photoreactions. Placing the cells in a medium of intermediate osmolarity produced a “chloroplast” preparation which maintained some capacity for O₂ evolution and CO₂ fixation, while possessing the ability to catalyze the photoinduced reduction of ferricyanide. Enzymic and photosynthetic reactions of cell-free preparations from D. parva were inhibited, rather than stimulated, by the salt concentration optimal for growth. These results were interpreted as indicating the existence of a steep NaCl gradient in vivo between the medium and the cell compartments which are not permeable to salt.     

盐胁迫对沙拐枣光合生理特性的影响

以采自甘肃民勤一年生的沙拐枣幼苗为试材,对不同NaCl浓度(0、50、100、200、300mmol·L~(-1))处理下沙拐枣光合生理特性进行分析,并对各生理指标与地上生物量进行灰色关联度分析,以探讨荒漠植物沙拐枣的抗盐机理,为沙拐枣的保护及其恢复荒漠生态系统稳定提供理论依据。结果显示:随着NaCl浓度的升高,沙拐枣同化枝内脯氨酸含量逐渐增大,而其可溶性糖含量逐渐减小;在低浓度NaCl(50mmol·L~(-1) NaCl)处理下,同化枝光合参数均增加,且净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)均达到最大值,比对照分别显著增加了33.3%、68.0%、60.8%;与50mmol·L~(-1) NaCl相比,处理浓度超过50mmol·L~(-1) NaCl时,Pn、Gs、Tr均降低;同化枝叶绿素b含量随着NaCl浓度的增加而降低,而叶绿素a和总叶绿素含量均呈先增加后降低的趋势。灰色关联度分析发现,同化枝的Tr、Gs、Ci以及叶绿素b与地上生物量的关联度较大。研究表明,低盐浓度NaCl激活了沙拐枣的某些生理机制,有利于植株的光合作用和生长,而植物在高盐浓度胁迫时能通过调节脯氨酸和可溶性糖的含量,减少叶绿素含量、Pn和Tr等维持自身的生长。     

Salt-Induced Metabolic Changes in Dunaliella salina

An increase of medium NaCl concentration induces Dunaliella cells to evolve O₂ photosynthetically even in the absence of CO₂. This NaCl-induced O₂ evolution may reflect the induced conversion of reserve carbohydrate to glycerol. The quantum yield for the NaCl-induced O₂ evolution, in the absence of CO₂, is 1.5-fold higher than that obtained for CO₂ fixation. Since the synthesis of glycerol from reserve carbohydrate in the absence of CO₂ requires only 0.5 ATP/NADPH, whereas photosynthesis requires at least 1.3 ATP/NADPH, it is concluded that the ATP/2e⁻ ratio coupled to NADP reduction in Dunaliella is lower than required for CO₂ fixation.     

Photosynthesis and photosystem 2 efficiency of two salt-adapted halophytic seashore <Emphasis Type="Italic">Cakile maritima</Emphasis> ecotypes

The effects of salinity (0–400 mM NaCl, marked S₀, S₁₀₀, S₂₀₀, and S₄₀₀) on growth, photosynthesis, photosystem 2 (PS2) efficiency, ion relations, and pigment contents were studied in two seashore Cakile maritima ecotypes (Tabarka and Jerba, respectively, sampled from humid and arid bioclimatic areas). Growth of Jerba plants was improved at S₁₀₀ as compared to S₀. Tabarka growth was inhibited by salinity at all NaCl concentrations. Leaf sodium and chloride concentrations increased with medium salinity and were higher in Jerba than in Tabarka plants. Chlorophyll content, net photosynthetic rate, stomatal conductance (g _s), and intracellular CO₂ concentration were stimulated at moderate salinity (S₁₀₀) in Jerba plants and inhibited at higher salt concentrations in both ecotypes: g _s was the most reduced parameter. The maximum quantum efficiency of PS2 (F_v/F_m), quantum yield, linear electron transport rate, and efficiency of excitation energy capture by open PS2 reaction centres showed no significant changes with increasing salt concentration in Jerba plant and were decreased in Tabarka subjected to S₄₀₀. However, the efficiency of dissipation of excess photon energy in the PS2 antenna was maintained in Jerba and was increased in Tabarka plants challenged with S₄₀₀. Hence the relative salt tolerance of Jerba was associated with a better ability to use Na⁺ and Cl⁻ for osmotic adjustment, the absence of pigment degradation, and the concomitant PS2 protection from photodamage.     

NaCl stress inhibits maltose fermentation by Saccharomyces cerevisiae

While fermentation of 20 g glucose l^–1 by Saccharomyces cerevisiae was not impaired by high NaCl concentrations, fermentation of 20 g maltose l^–1 was significantly decreased by 0.7 M NaCl, and completely inhibited with 1.4 M NaCl. No glycerol was produced in response to the salt stress when yeast cells were fermenting maltose. Active maltose transport, and not intracellular hydrolysis, was the metabolic step severely impaired by the NaCl stress.     

四翅滨藜生理生化特征对盐胁迫的响应

采用温室盆栽试验研究四翅滨藜(Atriplex canescens)幼苗株高、地径、生物量、净光合速率、蒸腾速率、气孔导度、叶绿素含量、抗氧化酶活性及丙二醛含量对不同浓度NaCl和Na_2SO_4(0、100、200、300和400mmol·L~(-1))胁迫的响应,以探讨四翅滨藜对不同种类及不同浓度盐渍环境的适应机制及其耐盐机理。结果显示:(1)随着盐分浓度的升高,四翅滨藜幼苗的株高、地径及生物量增量呈现出先升高后降低的趋势,低盐浓度下2种盐均促进幼苗生长,盐浓度超过400mmol·L~(-1)时,NaCl对幼苗生长具有明显抑制作用。(2)2种盐处理下,四翅滨藜幼苗净光合速率(Pn)和叶绿素含量(Chl)随盐浓度增大而升高,即2种盐均对幼苗Pn和Chl含量具有促进作用,且Na_2SO_4的促进效果大于NaCl;而幼苗蒸腾速率(Tr)和气孔导度(Gs)随盐浓度升高呈先增大后减小的趋势,且Na_2SO_4的促进作用强于NaCl。(3)与对照相比,四翅滨藜幼苗的丙二醛、SOD、POD酶活性在NaCl和Na_2SO_42种盐处理下,随着盐浓度的升高均呈现出不同程度的增大,且增大幅度总体表现为NaClNa_2SO_4。研究表明,四翅滨藜在NaCl和Na_2SO_4胁迫下,叶绿素的分解速率以及发挥作用的渗透调节物质均有差异,使得幼苗叶片健康程度不同,导致叶片光合能力大小的差异,最终表现为植株的生长差异;四翅滨藜具有较强的耐盐能力,而且对Na_2SO_4的适应能力强于NaCl。     

Effect of Ca2+ on the retention of glycerol and amino acids in Dunaliella tertiolecta

When D. tertiolecta cells, previously incubated in a 0.5 kmol m⁻³ NaCl medium with 1mol m⁻³ Ca²⁺, were transferred to an isotonic NaCl medium without Ca²⁺, the intracellular glycerol, as well as intracellular amino acids, was transiently lost to the medium within 30 min. The transient leakage of glycerol and amino acids was enhanced by the addition of EGTA (1 mol m⁻³), while the addition of SrCl₂ (1 mol m ⁻³) or polyamines such as spermidine (5 mol m⁻³) and spermine (5 mol m⁻³) restrained the leakage caused by the lack of external Ca²⁺ of intracellular glycerol and amino acids.     

Regulation of intracellular level of Na+, K+ and glycerol in Saccharomyces cerevisiae under osmotic stress

The intracellular level of Na⁺ and K⁺ of S. cerevisiae strain AB1375 revealed that under KCl as well as sorbitol stress, the cationic level was comparable to the level under no stress conditions. On the other hand, there was a sharp drop in the intracellular K⁺ content and increase in the Na⁺ content on addition of NaCl to the medium. However, the total cationic level was close to that under control conditions. In addition to changes in the cationic level, an enhanced production and accumulation of glycerol were also observed under osmotic stress. A regulatory mechanism co-ordinating the intracellular concentration of glycerol as well as Na⁺, K⁺ content under osmotic stress conditions has been proposed.     

Effect of pH, light intensity, salt and nitrogen concentrations on growth and β-carotene accumulation by a new isolate of Dunaliella sp

Summary In the present study Dunaliella sp. that could grow in the Johnson medium was isolated from hypersaline Lake Tuz and its β-carotene production was studied in a batch system, in order to determine the optimal conditions required for the highest β-carotene accumulation. In the experiments with light intensity, the cell numbers and β-carotene content were maximum at pH 9, with 20% of NaCl concentration and 48 kerg cm⁻² s⁻¹ light intensity. At this light intensity, the β-carotene content of Dunaliella sp. ranged between 0.177 and 1.095 mg/ml for the culture broth and 0.119 to 0.261 ng/cell on a per cell basis under the nitrogen limitation. At the end of the experiments, the maximum β-carotene accumulation and the cell number were obtained at pH 7, 5 mM NaNO₃ and 20% NaCl concentrations as 0.261 ng/cell, 4.2×10⁶ cell/ml, respectively.     

The Adaptation of Dunaliella to Widely-Differing Salt Concentrations

Methods are described for adapting pure cell-lines of Dunaliella,a green unicellular alga, to grow at concentrations between0.5 M and 3.5 M NaCl. It is shown that, provided large abruptchanges are avoided, cells of the same cell-line can becomeadapted to grow over this wide range of salt concentration.Once adapted, cultures are able to continue growing at a steadyrate for many generations provided that the salt concentrationremains constant. Viability tests performed after abrupt changesin salt concentration have shown that survival is higher afterdownward than after upward changes (3.5 M to 0.5 M NaCl: 20%;0.5 M to 3.5 M NaCl: 0.01% in D. parva 19/9). When the changein concentration from 0.5 M to 3.5 M NaCl takes place over aperiod of 48 h viability approaches 100%. The time needed for100% survival for a downward change over the same range is only1.5 h. There is no evidence for a genetic difference betweencell-lines adapted to particular concentrations of salt andit is concluded that the so-called ‘halophilic’and ‘halotolerant’ strains are interchangeable.It seems likely that the difference between the two types isa matter of gene expression. Key words: Dunaliella, salt concentration, viability