Polyphosphate Hydrolysis within Acidic Vacuoles in Response to Amine-Induced Alkaline Stress in the Halotolerant Alga Dunaliella salina

The location and mobilization of polyphosphates in response to an amine-induced alkaline stress were studied in the halotolerant alga Dunaliella salina. The following observations suggest that polyphosphates accumulate in acidic vacuoles: (a) Accumulation of large amounts of polyphosphates is manifested as intravacuolar dense osmiophilic bodies in electron micrographs. (b) Uptake of amines into the vacuoles induces massive hydrolysis of polyphosphates, demonstrated by in vivo ³¹P-nuclear magnetic resonance, and by analysis of hydrolytic products on thin layer chromatograms. The analysis indicates that: (a) Polyphosphate hydrolysis is kinetically correlated with amine accumulation and with the recovery of cytoplasmic pH. (b) The major hydrolytic product is tripolyphosphate. (c) The peak position of the tripolyphosphate terminal phosphate in nuclear magnetic resonance spectra is progressively shifted as the cells recover, indicating that the pH inside the vacuoles increases while the pH in the cytoplasm decreases. (d) In lysed cell preparations, in which vacuoles become exposed to the external pH, mild alkalinization in the absence of amines induces polyphosphate hydrolysis to tripolyphosphates. It is suggested that amine accumulation within vacuoles activates a specific phosphatase, which hydrolyzes long-chain polyphosphates to tripolyphosphates. The hydrolysis increases the capacity of the vacuoles to sequester amines from the cytoplasm probably by releasing protons required to buffer the amine, and leads to recovery of cytoplasmic pH. Thus, polyphosphate hydrolysis provides a high-capacity buffering system that sustains amine compartmentation into vacuoles and protects cytoplasmic pH.     

Ion Content of the Halotolerant Alga Dunaliella salina

The intracellular concentration of the major ions in Dunaliellasalina cells were determined, following the removal of extracellularions by ion-exchange minicolumns. Log phase cells, grown inmedia containing 1–4 molar NaCl, contained 30–50mM chloride and 200–350 mM magnesium (5 mM in medium).Phosphorus, which is present intracellularly mostly as polyphosphate,was present in amounts of 60–100 fmoles per cell, equivalentto a concentration of 600–1,000 mM (0.2 mM in medium).Previous data indicated that such cells contained 20–40mM Na⁺, 150–300mM K⁺, 20mM SO^2–₄, and very low concentrationsof Ca2⁺ and charged nitrogenous compounds. Mg²⁺ and K⁺ seemto serve as the major counter ions for the intracellular negativecharge present in the massively accumulated polyphosphates.The former accounts for about 2/3 of the required positive charge.This is supported by the observation that limitation in thephosphate or K⁺ supply in the medium lead to a parallel decreasein the accumulation of intracellular phosphorus, Mg²⁺ or K⁺. ¹Present address: Department of Vegetables, The Volcani Center,Bet-Dagan 50250, Israel. (Received June 13, 1988; Accepted August 25, 1988)     

Fatty Acid Acylated Proteins of the Halotolerant Alga Dunaliella salina

The unicellular, wall-less alga Dunaliella salina has been shown to contain an array of proteins modified by the covalent attachment of fatty acids. Myristic acid (14:0) comprised approximately 80% by weight of the protein-linked acyl groups in samples derived from cells cultured in medium containing 1.7 molar NaCl and 93% in samples from cells grown in medium containing 3.0 molar NaCl. Palmitic and stearic acids accounted for most of the remaining protein-bound acyl chains. Approximately 0.2% of the incorporated radioactivity was estimated to be in linkage with protein. The bulk of acyl chains (about 99%) were resistant to cleavage by alkali, indicating a preponderance of amide bonding. The sodium dodecyl sulfate-polyacrylamide electrophoresis labeling pattern of proteins from [³H]myristic-labeled cells was significantly different from that of proteins from cells exposed to [³H]palmitate. The appearance of radioactivity in certain proteins was also influenced by the salinity of the culture medium. Thus growth in moderate (1.7 molar) salt favored the acylation of a 48-kilodalton polypeptide whereas in high (3.0 molar) salt, a 17-kilodalton polypeptide was more heavily labeled.     

Determination of Ion Content and Ion Fluxes in the Halotolerant Alga Dunaliella salina

A method to determine intracellular cation contents in Dunaliella by separation on cation-exchange minicolumns is described. The separation efficiency of cells from extracellular cations is over 99.9%; the procedure causes no apparent perturbation to the cells and can be applied to measure both fluxes and internal content of any desired cation. Using this technique it is demonstrated that the intracellular averaged Na⁺, K⁺, and Ca²⁺ concentrations in Dunaliella salina cultured at 1 to 4 molar NaCl, 5 millimolar K⁺, and 0.3 millimolar Ca²⁺ are 20 to 100 millimolar, 150 to 250 millimolar, and 1 to 3 millimolar, respectively. The intracellular K⁺ concentration is maintained constant over a wide range of media K⁺ concentrations (0.5-10 millimolar), leading to a ratio of K⁺ in the cells to K⁺ in the medium of 10 to 1,000. Severe limitation of external K⁺, induces loss of K⁺ and increase in Na⁺ inside the cells. The results suggest that Dunaliella cells possess efficient mechanisms to eliminate Na⁺ and accumulate K⁺ and that intracellular Na⁺ and K⁺ concentrations are carefully regulated. The contribution of the intracellular Na⁺ and K⁺ salts to the total osmotic pressure of cells grown at 1 to 4 molar NaCl, is 5 to 20%.     

Na-NMR Studies of the Intracellular Sodium Ion Concentration in the Halotolerant Alga Dunaliella salina

The Intracellular Na⁺ concentration in the halotolerant alga Dunaliella salina was measured in intact cells by ²³Na-NMR spectroscopy, utilizing the dysprosium tripolyphosphate complex as a sodium shift reagent, and was found to be 88 ± 28 millimolar. Intracellular sodium ion content and intracellular volume were the same, within the experimental error, in cells adapted to grow in media containing between 0.1 and 4.0 molar NaCl. These values assume extracellular and intracellular NMR visibilities of the ²³Na nuclei of 100 and 40%, respectively. The relaxation rate of intracellular sodium was enhanced with increasing salinity of the growth medium, in parallel to the intracellular osmosity due to the presence of glycerol, indicating that Na⁺ ions and glycerol are codistribbuted within the cell volume.     

Partial Characterization of K and Ca Uptake Systems in the Halotolerant Alga Dunaliella salina

The uptake of K⁺ and Ca²⁺ in Dunaliella salina is mediated by two distinct carriers: a K⁺ carrier with a high selectivity against Na⁺, Li⁺, and choline⁺ but not towards Rb⁺, K⁺, Cs⁺, or NH₄⁺, and a Ca²⁺ carrier with a high selectivity against Mg²⁺. The latter is specifically blocked by La³⁺ and by Cd²⁺. Apparent K_m values for K⁺ and Ca²⁺ uptake are 2.5 and 0.8 millimolar, respectively, and their maximal calculated fluxes are 22 and 0.8 nanomoles per square meter per second, respectively. Effects of permeable ions and ionophores on K⁺ and Ca²⁺ uptake suggest that the driving force for their uptake is the transmembrane electrical potential. Inhibitors of ATP production, typical inhibitors of plasma membrane H⁺-ATPases and protonionophores inhibit K⁺ and Ca²⁺ uptake and accelerate K⁺ efflux. The results suggest that an H⁺-ATPase in the cell membrane provides the driving force for K⁺ and Ca²⁺ uptake. Efflux measurements from ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ loaded cells suggest that part of the intracellular K⁺ and most of the intracellular Ca²⁺ is nonexchangeable with the extracellular pool. Correlations between phosphate and K⁺ contents and the effect of phosphate on K⁺ efflux suggest intracellular associations between K⁺ and polyphosphates. On the basis of these results, it is suggested that: (a) K⁺ and Ca²⁺ uptake in D. salina is driven by the transmembrane electrical potential which is generated by the action of an H⁺-ATPase of the plasma membrane. (b) Part of the intracellular K⁺ is associated with polyphosphate bodies, while most of the intracellular Ca²⁺ is accumulated in intracellular organelles in the algal cells.     

Identification of Low Molecular Mass GTP-Binding Proteins in Membranes of the Halotolerant Alga Dunaliella salina

A family of specific guanine nucleotide-binding proteins in Dunaliella salina was studied. Polypeptides of different subcellular fractions were separated by electrophoresis and transferred to nitrocellulose or Immobilon membranes. Incubation of the transfer blots with [³⁵S]GTPγS or [α-³²P]GTP showed no evidence for GTP-binding proteins in the chloroplast and cytosol fractions. However, two GTP-binding proteins with molecular masses of 28 and 30 kilodaltons were present in the plasma membrane and microsomal fractions. An additional 29 kilodalton GTP-binding protein was detected in the plasma membrane. The mitochondrial fraction contained significant amounts of only the 28 kilodalton GTP-binding protein. Binding of [³²P]GTP to the protein blots was completely prevented by 10 micromolar GTP or guanosine 5′-O-(2-thiodiphosphate) (added in 3 × 10⁴-fold excess), whereas ATP or CTP had no effect on the binding. The 28 kilodalton GTP-binding protein was recognized by polyclonal antibodies to the ras-related YPT1 protein of yeast but not by the anti-ras Y13-259 monoclonal antibody. GTP-binding proteins present in the microsomal fraction could not be solubilized by incubation of microsomes with 1 molar NaCl or 0.2 molar Na₂CO₃, but some GTP-binding activity was solubilized when microsomes were treated with 6 molar urea. These results indicate that D. salina GTP-binding proteins are tightly associated with the membranes. The covalent attachment of fatty acids to these proteins was also investigated. Electrophoresis followed by fluorography of delipidated microsomal proteins extracted from [³H]myristic acid-labeled cells showed an intense labeling of a 28 kilodalton protein. We conclude that D. salina contains proteins resembling the ras-related proteins found in animal cells and higher plants.     

On the Factors Which Determine Massive beta-Carotene Accumulation in the Halotolerant Alga Dunaliella bardawil

Dunaliella bardawil, a β-carotene-accumulating halotolerant alga, has been analyzed for the effect of various growth conditions on its pigment content, and compared with Dunaliella salina, a β-carotene nonaccumulating species. In D. bardawil, increasing light intensity and light period or inhibiting growth by various stress conditions such as nutrient deficiency or high salt concentration caused a decrease in the content of chlorophyll per cell and an increase in the amount of β-carotene per cell. As a result, the β-carotene-to-chlorophyll ratio increased from about 0.4 to 13 grams per gram and the alga changed its visual appearance from green to deep orange. D. salina grown similarly decreased in content of both chlorophyll and β-carotene per cell and the culture turned from green to yellowish. Low chlorophyll-containing cells of D. bardawil or D. salina exhibit very high photosynthetic rates when expressed on a chlorophyll basis (~600 micromoles O₂ evolved per milligram chlorophyll per hour).

Variation of pigment content in D. bardawil by a large variety of environmental agents has been correlated with the integral irradiance received by the algal culture during a division cycle. The higher the integral irradiance per division cycle, the lower the chlorophyll content per cell; the higher the β-carotene content per cell, and therefore the higher the β-carotene-to-chlorophyll ratio. The results are interpreted as indicating a protecting effect of β-carotene against injury by high irradiance under conditions of impairment in chlorophyll content per cell.

     

In Vivo pH Regulation by a Na/H Antiporter in the Halotolerant Alga Dunaliella salina

Na⁺/H⁺ exchange activity in whole cells of the halotolerant alga Dunaliella salina can be elicited by intracellular acidification due to addition of weak acids at appropriate external pH. The changes in both intracellular pH and Na⁺ were followed. Following a mild intracellular acidification, intracellular Na⁺ content increased dramatically and then decreased. We interpret the phase of Na⁺ influx as due to the activation of the plasma membrane Na⁺/H⁺ antiporter and the phase of Na⁺ efflux as due to an active Na⁺ extrusion process. The following observations are in agreement with this interpretation: (a) the Na⁺ influx phase was sensitive to Li⁺, which is an inhibitor of the Na⁺/H⁺ antiporter, did not require energy, and was insensitive to vanadate; (b) the Na⁺ efflux phase is energy-dependent and sensitive to the plasma membrane ATPase inhibitor, vanadate. Following intracellular acidification, a drastic decrease in the intracellular ATP content is observed that is reversed when the cells regain their neutral pH value. We suggest that the intracellular acidification-induced change in the internal Na⁺ concentration is due to a combination of Na⁺ uptake via the Na⁺/H⁺ antiporter and an active, ATPase-dependent, Na⁺ extrusion. The Na⁺/H⁺ antiporter seems, therefore, to play a principal role in internal pH regulation in Dunaliella.     

P and C-NMR Studies of the Phosphorus and Carbon Metabolites in the Halotolerant Alga, Dunaliella salina

The intracellular phosphorus and carbon metabolites in the halotolerant alga Dunaliella salina adapted to different salinities were monitored in living cells by ³¹P- and ¹³C-nuclear magnetic resonance (NMR) spectroscopy. The ¹³C-NMR studies showed that the composition of the visible intracellular carbon metabolites other than glycerol is not significantly affected by the salinity of the growth medium. The T₁ relaxation rates of the ¹³C-glycerol signals in intact cells were enhanced with increasing salinity of the growth medium, in parallel to the expected increase in the intracellular viscosity due to the increase in intracellular glycerol. The ³¹P-NMR studies showed that cells adapted to the various salinities contained inorganic phosphate, phosphomonoesters, high energy phosphate compounds, and long chain polyphosphates. In addition, cells grown in media containing up to 1 molar NaCl contained tripolyphosphates. The tripolyphosphate content was also controlled by the availability of inorganic phosphate during cell growth. Phosphate-depleted D. salina contained no detectable tripolyphosphate signal. Excess phosphate, however, did not result in the appearance of tripolyphosphate in ³¹P-NMR spectra of cells adapted to high (>1.5 molar NaCl) salinites.     

A 150 Kilodalton Cell Surface Protein Is Induced by Salt in the Halotolerant Green Alga Dunaliella salina

Dunaliella salina is an extremely halotolerant, unicellular, green alga lacking a rigid cell wall. Osmotic adaptation to high salinities is based on the accumulation of glycerol. To uncover other functions responsible for halotolerance, protein profiles of algae continuously grown in different salinities were compared. A 150 kilodalton protein (p 150) increased in amount with salt concentration. Furthermore, when the cells were subjected to drastic hyperosmotic shocks, p150 started to rise long after completion of the osmotic response but coincident with reinitiation of cell proliferation. Cells with an initially higher level of p150 resumed growth faster than cells with a lower level of the protein. Addition of cycloheximide early after hyperosmotic shock prevented the rise in p150, indicating this rise was due to de novo synthesis of the protein. These observations suggest that p150 is a saltinduced protein required for proliferation of the cells in saline media. p150 was purified to homogeneity and found to be a detergent-soluble glycoprotein. Polyclonal antibodies against p150 recognized a single protein component in D. salina crude extracts. A high M_r cross-reacting protein was also observed in another Dunaliella strain, D. bardawil. Immunoelectron microscopy localized p150 to the cell surface.     

Modulation of Na/H Antiporter Activity by Extreme pH and Salt in the Halotolerant Alga Dunaliella salina

The effect of different growth conditions on the activity of the Na⁺/H⁺ antiporter in Dunaliella salina has been investigated. Adaptation of D. salina cells to ammonia at alkaline pH or to high NaCl concentrations is associated with a pronounced increase in the plasma membrane Na⁺/H⁺ exchange activity. The enhanced activity is manifested both in vivo, by stimulation of Na⁺ influx into intact cells in response to internal acidification, and in vitro, by a larger ²²Na accumulation in plasma membrane vesicles in response to an induced pH gradient. Kinetic analysis shows that the stimulation does not result from a change of the K_m for Na⁺ but from an increase in the V_max. In contrast, adaptation of cells to a high LiCl concentration (0.8 m) depresses the activity of the Na⁺/H⁺ antiporter. Adaptation to ammonia is also associated with a large increase of three polypeptide bands in purified plasma membrane preparations, indicating that they may compose the antiporter polypeptides. These results suggest that adaptation to ammonia or to high salinity induces overproduction of the plasma membrane Na⁺/H⁺ antiporter in Dunaliella.     

Salt-dependent Protein Composition in the Halotolerant Green Alga,Dunaliella parva

The extremely halotolerant green alga, Dunaliella parva, tolerates salt concentrations from 0.3 to 3.0 M NaCl. Effects of long-term adaptation to five distinct salinities were analyzed. Salt-dependent differences of physiological parameters such as growth rate, pigments, quantitative protein contents, and gas exchange were measured; furthermore the qualitative protein composition in salt-adapted cells was investigated using SDS-polyacrylamide gel electrophoresis. Proteins of apparent molecular masses of 26, 35, 39, 50, and 63 kDa were induced or intensified with an increase in external sodium chloride concentration whereas proteins of 85 and 101 kDa were diminished in high salt algae. After selective staining, four modifications of glycoproteins were observed. A glycoprotein of 96 kDa was produced exclusively in low salt cells whereas glycosylations of 105, 135, and 260 kDa were induced by high salt concentrations.     

Dynamics of Photosystem II and Its Light Harvesting System in Response to Light Changes in the Halotolerant Alga Dunaliella salina

A photosystem two (PSII) core complex consisting of five major polypeptides (47, 40, 32, 30, and 10 kilodaltons) and a light harvesting chlorophyll a/b complex (LHC-2) have been isolated from the halotolerant alga Dunaliella salina. The chlorophyll and polypeptide composition of both complexes were compared in illuminated and dark-adapted cultures. Dark adaptation is accompanied by a decrease in the chlorophyll a to chlorophyll b (Chl a/Chl b) ratio of intact thylakoids without any change in total chlorophyll. These changes occur with a half-time of 3 hours and are reversed upon reillumination. Analyses of PSII enriched membrane fragments suggest that the decrease in the Chl a/Chl b is due partly to an increase in the Chl b content of LHC-2 and partly to changes in the relative levels of the two complexes. Apparently during dark adaptation there is: (a) a net synthesis of chlorophyll b, (b) removal of PSII core complexes resulting in a 2-fold drop in the PSII cores to LHC-2 chlorophyll ratio. These changes should dramatically increase the light harvesting capacity of the remaining PSII reaction centers. Presumably this adjustment of antenna size and composition is a physiological mechanism necessary for responding to shade conditions. Also detected, using ³²P, are light-induced phosphorylation of the LHC-2 (consistent with the ability to undergo State transitions) and of the 40 and 30 kilodalton subunits of the PSII core complex. These observations indicate that additional mechanisms may also exist to help optimize the interception of quanta during rapid changes in illumination conditions.     

杜氏盐藻GAPDH cDNA的克隆鉴定及其启动子功能表达载体的构建

根据藻类甘油醛3-磷酸脱氢酶(Glyceraldehyde-3-phosphate dehydrogenase,GAPDH)氨基酸高度保守序列设计简并引物,采用5',3'-RACE和巢式PCR的方法,得到了杜氏盐藻(Dunaliella salina)GAPDH cDNA全长序列.序列同源性比较和进化树等生物信息学分析结果表明,根据获得的盐藻GAPDH的核酸序列推导的氨基酸序列与其他已知物种的GAPDH有较高的同源性.定向克隆于原核表达载体的盐藻GAPDH cDNA在大肠杆菌中以融合蛋白的形式得到了高效表达,并成功构建了由盐藻GAPDH基因启动子驱动的cat(氯霉素乙酰转移酶)基因表达载体pUCGCat,为进一步研究杜氏盐藻GAPDH基因和启动子功能奠定了试验基础.     

杜氏盐藻细胞质膜氧化还原系统

杜氏盐藻细胞质膜具有氧化ＮＡＤ（Ｐ）Ｈ、还原Ｆｅ（ＣＮ）和Ｏ２的氧化还原系统。当Ｆｅ（ＣＮ）浓度为０．６ｍｍｏｌ／Ｌ时,氧化ＮＡＤＨ的Ｋｍ为９６μｍｏｌ／Ｌ,Ｔｍａｘ为１５９ｎｍｏｌ１０－８ｃｅｌｌｓｍｉｎ－１,最适ｐＨ为８．５。ＴｒｉｔｏｎＸ－１００可促进ＮＡＤＨ和Ｆｅ（ＣＮ）的氧化还原活性。ＮＡＤＨ能促进藻细胞的氧吸收,最适ＰＨ为８．５。在无外源电子供体存在时,细胞质电子供体提供的电子使Ｆｅ（ＣＮ）还原。培养液ＰＨ影响正常呼吸链、交替氧化酶途径和质膜电子传递链的耗氧比例;当有外源ＮＡＤＨ存在时,ＳＨＡＭ明显促进细胞的氧吸收,并且质膜电子传递链的耗氧比例增加。     

Plasma Membrane Sterols Are Essential for Sensing Osmotic Changes in the Halotolerant Alga Dunaliella

The halotolerant alga Dunaliella responds to hyperosmotic stress by synthesis of massive amounts of glycerol. The trigger for this osmotic response is the change in cell volume, but the mechanism that senses volume changes is not known. Preincubation of Dunaliella salina with tridemorph, a specific inhibitor of sterol biosynthesis, inhibits glycerol synthesis and volume recovery. The inhibition is associated with suppression of [14C]bicarbonate incorporation into sterols and is correlated with pronounced depletion of plasma membrane sterols. Incubation of sterol-depleted cells with cholesterol hemisuccinate restores the capacity for volume regulation in response to hyperosmotic stress. Tridemorph as well as lovastatin also inhibit volume changes that are induced by high light in Dunaliella bardawil, a species that responds to high light intensity by synthesis of large amounts of [beta]-carotene. These volume changes result from accumulation of glycerol and are associated with de novo synthesis of sterols. The major plasma membrane sterol in D. salina and the high-light-induced sterol in D. bardawil co-migrate with ergosterol on thin-layer chromatography and on reversed-phase high-performance liquid chromatography. These results suggest that the osmosensory mechanism in Dunaliella resides in the plasma membrane, and that sterols have an important role in sensing osmotic changes.     

Transcriptome Profiling of the Salt-Stress Response in the Halophytic Green Alga Dunaliella salina

Plant Molecular Biology Reporter - Due to the hypersaline environment cell of Dunaliella salina can change its morphology, growth, and pigment for adapting to the stress. Despite the fact D. salina...     

杜氏盐藻质膜ATPase在渗透调节中的可能作用

杜氏盐藻是一种以甘油为渗透调节物质的单细胞海藻,能够在0.08～5.0mol/L NaGl的培养液中生长。当外界NaGl浓度从0.5mol/L上升到4.0mol/L时,藻细胞内的Na~+和K~+含量变化不大,甘油含量则从6.20Pg/cell上升到51.50pg/cell。当藻细胞承受2.0mol/L到3.0mol/L NaCl的高渗胁迫时,能通过增加细胞内甘油含量来恢复原有形态;同时,藻细胞的H~+分泌增加,ATP含量下降;20μmol/L Na_3VO_4抑制了这些变化。KGN处理虽降低藻细胞内的ATP含量,却增加K~+外流和Na~+内渗。