Halotolerant Green Alga Dunaliella Research Articles

Triacylglycerol is produced from starch and polar lipids in the green alga Dunaliella tertiolecta.

The halotolerant green alga Dunaliella tertiolecta accumulates starch and triacylglycerol (TAG) amounting to 70% and 10-15% of total cellular carbon, respectively, when exposed to nitrogen (N) deprivation. The purpose of this study was to clarify the inter-relationships between the biosynthesis of TAG, starch, and polar lipids (PLs) in this alga. Pulse labeling with [14C]bicarbonate was utilized to label starch and [14C]palmitic acid (PlA) to label lipids. Transfer of 14C into TAG was measured and used to calculate rates of synthesis. About two-thirds of the carbon in TAG originates from starch, and one-third is made de novo by direct CO2 assimilation. The level made from degradation of pre-formed PLs is estimated to be very small. Most of the de novo synthesis involves fatty acid transfer through PLs made during the first day of N deprivation. The results suggest that starch made by photosynthetic carbon assimilation at the early stages of N deprivation is utilized for synthesis of TAG. Trans-acylation from PLs is the second major contributor to TAG biosynthesis. The utilization of starch for TAG biosynthesis may have biotechnological applications to optimize TAG biosynthesis in algae.

Proteome analysis of cytoplasmatic and plastidic β-carotene lipid droplets in Dunaliella bardawil.

The halotolerant green alga Dunaliella bardawil is unique in that it accumulates under stress two types of lipid droplets: cytoplasmatic lipid droplets (CLD) and β-carotene-rich (βC) plastoglobuli. Recently, we isolated and analyzed the lipid and pigment compositions of these lipid droplets. Here, we describe their proteome analysis. A contamination filter and an enrichment filter were utilized to define core proteins. A proteome database of Dunaliella salina/D. bardawil was constructed to aid the identification of lipid droplet proteins. A total of 124 and 42 core proteins were identified in βC-plastoglobuli and CLD, respectively, with only eight common proteins. Dunaliella spp. CLD resemble cytoplasmic droplets from Chlamydomonas reinhardtii and contain major lipid droplet-associated protein and enzymes involved in lipid and sterol metabolism. The βC-plastoglobuli proteome resembles the C. reinhardtii eyespot and Arabidopsis (Arabidopsis thaliana) plastoglobule proteomes and contains carotene-globule-associated protein, plastid-lipid-associated protein-fibrillins, SOUL heme-binding proteins, phytyl ester synthases, β-carotene biosynthesis enzymes, and proteins involved in membrane remodeling/lipid droplet biogenesis: VESICLE-INDUCING PLASTID PROTEIN1, synaptotagmin, and the eyespot assembly proteins EYE3 and SOUL3. Based on these and previous results, we propose models for the biogenesis of βC-plastoglobuli and the biosynthesis of β-carotene within βC-plastoglobuli and hypothesize that βC-plastoglobuli evolved from eyespot lipid droplets.

Phytoplankton assemblage of a solar saltern in Port Fouad, Egypt

The present study is the first investigation of the phytoplankton community in one of Egypt’s saltworks. The phytoplankton composition and distribution in five ponds of increasing salinity were investigated in the solar saltern of Port Fouad. The phytoplankton community consisted of 42 species belonging to cyanobacteria (16), diatoms (12), dinoflagellates (11), Euglenophyceae (2) and Chlorophyceae (1). The number of species decreased significantly and rapidly with increasing salinity, varying between 33 species in the first pond (P1) and one species in the crystallizer pond (P5). Conversely, the total phytoplankton density, except that recorded in P1, increased significantly with rising salinity, fluctuating between 8.7 and 56×105 individuals l−1 in P2 and P5 respectively. In spite of the local variations in climate and nutrient availability, the phytoplankton composition, density and spatial variations along the salinity gradient were, in many respects, very similar to what has been observed in other solar saltworks. The pond with the lowest salinity (P1−< 52gl−1) was characterized by a significant diversity and blooming of diatoms and dinoflagellates. Intermediate salinity ponds (P2 and P3) with salinity∼112–180gl−1 exhibited a decline in both species richness and density, but the stenohaline blue green algae (Synechocystis salina) did flourish. The highly saline concentrating ponds and crystallizers (P4 and P5) with salinity∼223–340gl−1 were characterized by few species, the disappearance of blue green algae and the thriving of the halotolerant green alga Dunaliella salina.

Isolation and characterization of a stress-inducible Dunaliella salina Lcy-β gene encoding a functional lycopene β-cyclase

The halotolerant green alga Dunaliella salina accumulates large amounts of beta-carotene when exposed to various stress conditions. Although several studies concerning accumulation and biotechnological production of beta-carotene have been published, the molecular basis and regulation of the genes involved in carotenoid biosynthesis in D. salina are still poorly known. In this paper, we report the isolation and regulation of the lycopene beta-cyclase (Lcy-beta) gene by abiotic stress. The function of this gene was determined by heterologous genetic complementation in E. coli. Gene expression and physiological analyses revealed that D. salina Lcy-beta steady-state transcript and carotenoid levels were up-regulated in response to all stress conditions tested (salt, light and nutrient depletion). The results presented here suggest that nutrient availability is a key factor influencing carotenogenesis as well as carotenoid biosynthesis-related gene expression in D. salina.

Cloning and expression study of a putative carotene biosynthesis related (cbr) gene from the halotolerant green alga Dunaliella salina

Dunaliella salina, a unicelluar green alga that can tolerate an extreme variation of salt concentration is being studied as a model system to analyze the tolerance of abiotic stresses at the molecular level. Upon abnormal NaCl levels, new transcripts were abundantly expressed in cells of the alga. EST gene discovery efforts utilizing salt-shock cells had identified one cDNA designated Dscbr (GenBank accession no. DQ867041) with significant similarity to a carotene biosynthesis related gene (cbr) from Dunaliella bardawil and to early light inducible genes (elip) of higher plants. Dscbr was 976 bp in length, encoding a 190 amino acid deduced polypeptide (DsCBR) with a predicted molecular mass of 19.9 kDa and pI of 9.0. The three dimensional structure of DsCBR modeled by computer homology modeling techniques showed that the protein possessed three predicted transmembrane helices and six conserved pigment-binding residues. Real-Time Quantitative PCR clearly demonstrated that Dscbr mRNA can be rapidly induced by high light intensity and salt shocks. The results presented in this work are consistent with the earlier proposal (Jin et al. 2001 Biochim Biophys Acta 1506:244-259, 2003 Plant Physiol 132:352-364) that the DsCBR protein is an adaptive response to stress-induced photodamage within the alga chloroplast, and plays a key role in the protection and/or repair of the photosynthetic apparatus.

Valorization attempt of a Moroccan salt pond: -carotene production by the halotolerant green alga Dunaliella salina

Valorization attempt of a Moroccan salt pond: -carotene production by the halotolerant green alga Dunaliella salina

Enhanced photosynthesis and redox energy production contribute to salinity tolerance in Dunaliella as revealed by homology-based proteomics.

Salinity is a major limiting factor for the proliferation of plants and inhibits central metabolic activities such as photosynthesis. The halotolerant green alga Dunaliella can adapt to hypersaline environments and is considered a model photosynthetic organism for salinity tolerance. To clarify the molecular basis for salinity tolerance, a proteomic approach has been applied for identification of salt-induced proteins in Dunaliella. Seventy-six salt-induced proteins were selected from two-dimensional gel separations of different subcellular fractions and analyzed by mass spectrometry (MS). Application of nanoelectrospray mass spectrometry, combined with sequence-similarity database-searching algorithms, MS BLAST and MultiTag, enabled identification of 80% of the salt-induced proteins. Salinity stress up-regulated key enzymes in the Calvin cycle, starch mobilization, and redox energy production; regulatory factors in protein biosynthesis and degradation; and a homolog of a bacterial Na(+)-redox transporters. The results indicate that Dunaliella responds to high salinity by enhancement of photosynthetic CO(2) assimilation and by diversion of carbon and energy resources for synthesis of glycerol, the osmotic element in Dunaliella. The ability of Dunaliella to enhance photosynthetic activity at high salinity is remarkable because, in most plants and cyanobacteria, salt stress inhibits photosynthesis. The results demonstrated the power of MS BLAST searches for the identification of proteins in organisms whose genomes are not known and paved the way for dissecting molecular mechanisms of salinity tolerance in algae and higher plants.

Mitochondrial NDP kinase from Dunaliella tertiolecta (Chlorophyceae, Chlorophyta)

SUMMARY A cDNA ( DtNDK1 ) encoding a nucleoside diphosphate(NDP) kinase with a putative mitochondrial targetingsignal sequence was previously isolated from the halo-tolerant green alga Dunaliella tertiolecta . Whenexpressed in Saccharomyces cerevisiae , the processedDtNDK1 enzyme was specifically localized to mito-chondria. The present study reports several biochemi-cal characteristics of the mitochondrial NDP kinasefrom D. tertiolecta . The mature protein was expressedas either N- or C-terminal hexahistidine-tagged proteinand purified to homogeneity by affinity chromato-graphy. Native gel electrophoresis and sedimentationvelocity in sucrose density gradients showed that theactive enzyme consisted of a hexamer. The enzyme,with a pH optimum of 7, showed the highest specificityto dCDP ( K m = 50 µ mol/L) and the highest turnovertowards the synthesis of UTP (up to 140-fold higher).The present study also provides evidence that purifiedDtNDK1 proteins are capable of transferring a phos-phate group to another protein.Key words:

Photosynthetic acclimation to photon irradiance and its relation to chlorophyll fluorescence and carbon assimilation in the halotolerant green alga Dunaliella viridis.

This work describes the long-term acclimation of the halotolerant microalga Dunaliella viridis to different photon irradiance, ranging from darkness to 1500 mumol m(-2) s(-1). In order to assess the effects of long-term photoinhibition, changes in oxygen production rate, pigment composition, xanthophyll cycle and in vivo chlorophyll fluorescence using the saturating pulse method were measured. Growth rate was maximal at intermediate irradiance (250 and 700 mumol m(-2) s(-1)). The increase in growth irradiance from 700 to 1500 mumol m(-2) s(-1) did not lead to further significant changes in pigment composition or EPS, indicating saturation in the pigment response to high light. Changes in Photosystem II optimum quantum yield (F(v)/F(m)) evidenced photoinhibition at 700 and especially at 1500 mumol m(-2) s(-1). The relation between photosynthetic electron flow rate and photosyntetic O(2) evolution was linear for cultures in darkness shifting to curvilinear as growth irradiance increased, suggesting the interference of the energy dissipation processes in oxygen evolution. Carbon assimilation efficiencies were studied in relation to changes in growth rate, internal carbon and nitrogen composition, and organic carbon released to the external medium. All illuminated cultures showed a high capability to maintain a C:N ratio between 6 and 7. The percentage of organic carbon released to the external medium increased to its maximum under high irradiance (1500 mumol m(-2) s(-1)). These results suggest that the release of organic carbon could act as a secondary dissipation process when the xanthophyll cycle is saturated.

Metabolic response of the halotolerant green algaDunaliella bardawil to nitrogen: Phosphorus ratios in batch culture

The effect of N/P ratios and salinity on the growth as well as some metabolic activities by the halotolerant green algaDunaliella bardawil were followed. In nutrient-deficient media (N0 or P0) growth was retarded and cells accumulated large amounts of carotene, glycerol and saccharides while the contents of protein and amino acids were significantly decreased. In contrast, in nutrient-enriched media (N5 and/or P5), the growth and protein content in addition to the oxygen evolution were significantly increased at the expense of glycerol and saccharide content.

Activation of a 74 kDa plasma membrane protein kinase by hyperosmotic shocks in the halotolerant alga Dunaliella salina

The purpose of this study was to find out whether protein kinases are involved in the osmotic response of the halotolerant green alga Dunaliella salina . Hyperosmotic shock leads to a rapid transient activation of two different protein kinases, identified by an in-gel assay: A 40 kD kinase (PK 40), which is activated within seconds after osmotic shocks, and a 74 kD kinase (PK 74), activated a few minutes after the shock. Subcellular fractionation shows that PK 40 is a soluble enzyme whereas PK 74 is a membrane-associated protein localized in a plasma membrane-enriched microsomal fraction. Heat shock and cytoplasmic acidification activate a 40 kD, but not a 74 kD protein kinase. Both PK 40 and PK 74 phosphorylate histone at threonine residues and do not require calcium ions. The following results suggest that PK 74 may be a novel type of a MEK 1 kinase: 1) PK 74 phosphorylates ERK 1, a specific substrate of mammalian MEK 1, and 2). Antibodies against a conserved peptide of MEK 1 cross-react specifically with a 74 kD component localized at the plasma membrane fraction, which possesses the PK 74 activity. It is suggested that PK 74 is activated by osmotically-induced structural changes at the plasma membrane. Its possible role in the osmotic signal transduction is discussed.

The Effects of Sudden Salt stress on Protein Synthesis in the Green Alga Dunaliella parva

Summary Protein synthesis of the halotolerant green alga Dunaliella parva adapted to 0.75 and 1.5 M NaCl was compared with synthesis following a hyper- or hypoosmotic salt stress by using 35 S-protein-labelling and one- and two-dimensional polyacrylamide gel electrophoresis. Changes in salinity induced four types of modifications: Transient and permanent intensifications of proteins, and transient and permanent decreases. The majority of modifications occurred only during the first hours after the osmotic shock; both hyper- and hypoosmotic stress induced a number of analogous changes in protein synthesis. A few of these modifications could be still detected after long-term adaptation to the two different salt concentrations. Prominent differences were found, e.g. in proteins of 36 and 170 kDa, typically expressed in algae adapted to 1.5 M NaCl; these polypeptides were absent or synthesized at very low rates in cells of 0.75 M NaCl. Proteins of, e.g., 23, 24.5, and 37.5 kDa showed their highest rate of expression in the low salt algae.

Influence of areal density on β-carotene production by Dunaliella salina

High irradiance is probably the most important factor responsible for the massive accumulation of β-carotene by the halotolerant green alga Dunaliella salina. Operating outdoor cultures at optimal areal densities should result in maximal productivity. It is known that the optimal areal density is not fixed for all algae, where it could vary depending on the type of algae cultured, pond construction, turbulence and prevailing environmental conditions. At biomass concentrations below the optimum, more light per cell is available than that which could be absorbed by the biomass. These high light conditions should favour carotenogenesis and could result in higher β-carotene production rates. The results obtained clearly showed that over and above light and nutrient stress, an extremely important aspect is the residence time of the cells in the ponds. Longer residence times resulted in the development of larger cells, containing larger quantities of β-carotene. Productivity of biomass and β-carotene were about 70% higher at areal densities of 35–45 g m-2, compared to areal densities of 15–25 g m-2.

Effect of growth arrest on carotene accumulation and photosynthesis in Dunaliella

The halotolerant green alga Dunaliella bardawil is known to accumulate β-carotene in response to stress factors such as high light intensity, high salt concentrations and nutrient limitation. In this report, the accumulation of β-carotene was studied in cells from nitrate-limited chemostat cultures, in comparison with those of D. salina, a strain that does not accumulate β-carotene under stress conditions. D. bardawil responded to growth arrest by accumulating β-carotene and, to a lesser degree, lutein and zeaxanthin. A substantial fraction of β-carotene and all the lutein and zeaxanthin was associated with the thylakoid fraction. The accumulation of carotenoids in D. bardawil occurred only in the light, but the light intensities were far below those where the photosynthetic rate is maximal. After growth arrest, the amount of chlorophyll (Chi) decreased in both strains. However, in D. bardawil Chi a decreased to a lesser extent in comparison with Chi b, which resulted in an increased Chi alb ratio. The maximum photosynthetic capacity declined rapidly in both strains after growth arrest. In contrast, the photosynthetic efficiency showed a temporary increase in D. bardawil and a decrease in D. salina. This increase did not occur when carotenogenesis was inhibited by diphenylamine, implying a causal relationship between enhanced carotenogenesis and increase of photosynthetic efficiency. The possible involvement of stress-accumulated carotenoids in photosynthetic activity is discussed.

Ca 2+-dependent protein kinase from the halotolerant green alga Dunaliella tertiolecta: Partial purification and Ca 2+-dependent association of the enzyme to the microsomes

Ca 2+-dependent protein kinase (CDPK) was purified 900-fold from the soluble fraction of Dunaliella tertiolecta cells by ammonium sulfate precipitation, DEAE—Toyopearl, phenyl-Sepharose, and hydroxylapatite column chromatography. The CDPK was activated by micromolar concentration of Ca 2+ and required neither calmodulin nor phospholipids for its activation. The enzyme phosphorylated casein, myosin light chain, and histone type III-S (histone H-1), but did not phosphorylate protamine and phosvitin. The K m values for ATP and casein were 11 μ m and 300 μg/ml, respectively. Phosphorylation of casein was inhibited by calmodulin antagonists, calmidazolium, trifluoperazine, and compound 48 80 , but not affected by calmodulin. CDPK bound to phenyl-Sepharose in the presence of Ca 2+ and was eluted by ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA). This suggests that hydrophobicity of the enzyme was increased by Ca 2+. CDPK was also bound to the microsomes isolated from Dunaliella cells in the presence of micromolar concentration of Ca 2+ and released in the presence of EGTA, suggesting the possibility of in vivo Ca 2+-dependent association of the enzyme. The enzyme phosphorylated many proteins in the microsomes but few in the cytosol, if at all.

A mutant ofDunaliella parva CCAP 19/9 leaking large amounts of glycerol into the medium

A mutant of the halotolerant green algaDunaliella parva, which leaks large amounts of intracellular glycerol into the surrounding medium, was isolated. The mutant has potential applications in the commercial production of glycerol on a large scale since there is no need to extract glycerol from the cells. The mutant was compared with the wild type and it was found that, despite the leakage of glycerol, the mutant showed the same growth rate as the wild type. However, when the rates of oxygen evolution and uptake and intracellular starch content between mutant and wild type were compared at high salinity, considerable differences were found.

Abscisic Acid Metabolism in Salt-Stressed Cells of Dunaliella salina

The interrelationship between abscisic acid (ABA) production and beta-carotene accumulation was investigated in salt-stressed cells of the halotolerant green alga Dunaliella salina var bardawil. Cells were supplied with either R-[2-(14)C]mevalonolactone or [(14)C] sodium bicarbonate for 20 hours and then exposed to increased salinity (1.5 to 3.0 molar NaCl) for various lengths of time. Incorporation of label into abscisic acid and phaseic acid and the distribution of [(14)C]ABA between the cells and incubation media were monitored. [(14)C]ABA and [(14)C]phaseic acid were identified as products of both R-[2-(14)C]mevalonolactone and [(14)C]sodium bicarbonate metabolism. ABA metabolism was enhanced by hypersalinity stress. Actinomycin D, chloramphenicol, and cycloheximide abolished the stress-induced production of ABA, suggesting a role for gene activation in the process. Kinetic analysis of both ABA and beta-carotene production demonstrated two stages of accelerated beta-carotene production. In addition, ABA levels increased rapidly, and this increase occurred coincident with the early period of accelerated beta-carotene production. A possible role for ABA as a regulator of carotenogenesis in cells of D. salina is therefore discussed.

L-1-N-methyl-4-mercaptohistidine disulfide, a potential endogenous regulator in the redox control of chloroplast coupling factor 1 in Dunaliella.

A water-soluble, highly polar, heat-stable, small molecule has been isolated from cell-free extracts of the halotolerant green alga Dunaliella salina. This compound, soluble inhibitory factor (SIF), when added to in vivo light-activated, thylakoid membrane-bound preparations of the D. salina coupling factor 1 (CF1), causes a rapid inactivation of the ATPase activity. SIF must be in its oxidized form to inactivate the CF1 ATPase and probably functions by oxidizing the reduced form of the light-activated enzyme. SIF has been purified to homogeneity and characterized by UV-visible and IR absorption spectroscopy, 1H and 13C NMR spectroscopy, and mass spectrometry. SIF has five different kinds of nonexchangeable protons and seven different kinds of carbon atoms. Three of the carbon atoms and one proton are part of a heterocyclic (imidazole) ring. One carbon atom is a carbonyl (carboxylic acid). One carbon atom and three protons form a methyl group attached to the aromatic ring. One carbon atom and two protons are a methylene group, and one carbon atom (an alpha-amino carbon) is attached to a single proton. In addition, in its reduced form, SIF contains a thiol group attached to the heterocyclic ring. From high resolution mass spectrometry, the molecular weight of SIF was determined to be 401 (M + H+) and is consistent with the composition being C14H21N6O4S2. The UV absorption of SIF shows a large increase at 240 nm upon reduction. An effective difference extinction coefficient for this absorbance change has been calculated to be 6.84 meq/cm. A comparison of SIF with the oxidized form of ovothiol A (1-N-methyl-4-mercaptohistidine disulfide) shows the two compounds to be identical in all respects. In addition, ovothiol A disulfide is as effective as SIF in inhibiting the light-triggered, CF1 ATPase activity. It is concluded, therefore, that SIF and L-1-N-methyl-4-mercaptohistidine disulfide are identical.

Cloning and nucleotide sequence analysis of genes coding for the major chlorophyll-binding protein of the moss Physcomitrella patens and the halotolerant alga Dunaliella salina

Two clones have been isolated from a genomic library of the moss Physcomitrella patens and a cDNA library of the halotolerant green alga Dunaliella salina. The isolates contain genes coding for the major light-harvesting chlorophyll- a/ b-binding protein (CAB) in the photosystem II (PSII) light-harvesting complex (LHCII). The 2544-bp insert of the moss genomic clone contains the complete CAB-coding region and 5' and 3' flanking sequences. The coding region contains an intron of 359 bp which is spanned by a pair of 9-bp perfect direct repeats. There are two CCAAT boxes and five enhancer-like elements related to (G)TGG AAA TTT(G) (Weiher AAA et al., 1983) residing in the intron. Comparisons of the moss cab gene with sequences of light-inducible genes of higher plants reveal homologous and repeated sequences similar to the enhancer element in the 5' region upstream from the TATA and CCAAT boxes thought to be responsive to light inducibility. The 1256-bp algal cDNA contains the complete CAB-coding sequence, a 170-bp 5'-nontranslated region, and a 264-bp 3'-non-translated region. While the overall homology in the nontranslated regions is low between the cab gene of the moss and that of the alga, the 3'-nontranslated regions of the two contain some sequences that are conserved among the cab genes in higher plants. The deduced amino acid sequences of these two clones are highly conserved except for the N-terminal region. Their hydropathic plots are very similar and both possess three hydrophobic segments that are likely α-helical transmembrane segments. The proposed CAB transit peptide sequence of the alga is divergent from that of the moss or higher plants, suggesting that they may have evolved from different origins. Southern blot analysis shows that the cab genes in the moss and the alga, as in higher plants, are encoded by a number of homologous genes constituting a multigene family.

Localization of glycollate dehydrogenase in Dunaliella salina

Glycollate dehydrogenase of the halotolerant green alga Dunaliella salina, isolated from a brine pond, was found associated with the membrane fraction which exhibited complete photosynthetic activity. Highest enzyme activity was found in cells grown in the presence of 5% NaCl. Any increase in NaCl concentration led to a decrease in specific enzyme activity.