Photoperiod influences endogenous indoleamines in cultured green alga Dunaliella bardawil

Effect of light intensity and photoperiod on growth, indoleamines and carotenoid production was studied in unicellular green algae D. bardawil. Maximum biomass and carotenoid contents were found when cultures were grown in light (intensity of 2.0 Klux) at a photoperiod of 16/8h light and dark cycle. There was a profound influence of tested photoperiod conditions of light:dark viz. 8:16, 10:14, and 12:12 hr, continuous light on indoleamines (SER and MEL) production as estimated by HPLC and confirmed by mass spectral data obtained from LC-MS-ESI studies. Serotonin level increased from 908 to 1765 pg/g fresh wt with increase in light duration and melatonin level increased from 267 to 584 pg/g fresh wt during increase in dark phase. Carotenoids production was high in continuous light than other tested conditions.     

Bioavailability of the isomer mixture of phytoene and phytofluene-rich alga Dunaliella bardawil in rat plasma and tissues

Dunaliella bardawil, a beta-carotene-accumulating alga was treated by the bleaching herbicide norflurazon to select sub-species rich with a mixture of 9-cis and all-trans stereoisomers of phytoene and phytofluene. The present study determines the bioavailability of phytoene and phytofluene with their stereoisomers in rats fed on a diet supplemented with Dunaliella phytoene-rich spray dried powder. Three groups of female weanling rats, eight animals each, were fed AIN diets for two weeks. The control consumed the diet as is. The experimental group was supplemented with 50 g Dunaliella powder to give phytoene/phytofluene at a level of 1 g/kg diet, and the placebo was provided with the oxidized algae free of carotenoids at the same amount. Weight gain and tissues weight of rats fed on the control diet, or on the experimental diets were statistically same. Tissue analyses were carried out by liquid chromatography at the end of two weeks feeding for vitamin A, carotenoids, phytoene and phytofluene and theirs stereoisomers. Liver analyses revealed high hepatic storage of phytoene in the experimental group. Analysis of the other tissues, adrenal, brain, heart, kidney, lung, and spleen detected small amounts of phytoene in the adrenal, kidney and spleen and in the plasma. High-pressure liquid chromatography for stereoisomeric composition was performed to all phytoene-containing tissues. The original algal diet content of 9-cis-to-all-trans ratio of 1:1 was maintained in the plasma and adrenal while in the liver, spleen and kidney the ratio was reduced to 1:3. The preferential accumulation of all-trans phytoene over 9-cis phytoene in the liver, spleen and kidney may be interpreted as indicating stronger antioxidative effect of 9-cis phytoene over the all-trans isomer or alternatively, in vivo streoisomerization of 9-cis phytoene to the all-trans structure.     

Far-red light inhibits growth but promotes carotenoid accumulation in the green microalga Dunaliella bardawil

It has been demonstrated that far-red light reduces growth of marine phytoplankton and that light quality controls growth and photosynthetic metabolism in algae. The green halotolerant microalga, Dunaliella bardawil, accumulates high amounts of β-carotene (up to 10% of its dry weight) under conditions of high light or nutrient limitation. The influence of increasing irradiance and of far-red light in D. bardawil was studied. Continuous irradiance was provided by white fluorescent lamps alone (WL) or supplemented with far-red Linestra lamps (WL+FR). For both types of light, cultures were acclimatized at increasing irradiances (50-300 µmol m^?2 s^?1), and cell density, photosynthetic activity and pigment content were determined. Cell density increased with the photon irradiance, and was higher in WL than in WL+FR under the same irradiance, but the reverse occurred in respect of cell volume. Growth rate was higher under WL+FR. Far-red light induced faster growth but reduced the maximal cell density of the cultures. Chlorophyll a concentration was higher in white light, but total carotenoid content increased dramatically in both far-red light treatments (about 50% on a per cell basis) and with the increase of irradiance. Our results show that far-red light has a significant influence on growth and photosynthesis of D. bardawil, inducing a decrease in cell density, photosynthetic activity and chlorophyll concentration, and an increase in growth rate, cell volume and carotenoid content.     

Non-photochemical quenching of chlorophyll fluorescence in the green alga Dunaliella

The relaxation of the non-photochemical quenching of chlorophyll fluorescence has been investigated in cells of the green alga Dunaliella following illumination. The relaxation after the addition of DCMU or darkening was strongly biphasic. The uncoupler NH₄Cl induced rapid relaxation of both phases, which were therefore both energy-dependent quenching, qE. The proportion of the slow phase of qE increased at increasing light intensity. In the presence of the inhibitors rotenone and antimycin the slow phase of qE was stabilised for in excess of 15 min. NaN₃ inhibited the relaxation of almost all the qE. The implications of these results are discussed in terms of the interpretation of the non-photochemical quenching of chlorophyll fluorescence in vivo and the mechanism of qE.Abbreviations PS II Photosystem II - qQ photochemical quenching of chlorophyll fluorescence - qNP non-photochemical quenching of chlorophyll fluorescence - qE energy-dependent quenching of chlorophyll fluorescence - F _m maximum level of chlorophyll fluorescence for dark adapted cells - F _m level of fluorescence at any time when qQ is zero     

An actin-like protein and gene in the unicellular green alga Dunaliella

We present evidence for an actin-like protein in the unicellular alga Dunaliella (Volvocales) which has a molecular weight comparable to that of vertebrate skeletal muscle actin. Intracellular detection using indirect immunofluorescence indicates that this protein is present in the cytoplasm. Using mouse actin c-DNA probes, we show that this protein is probably encoded by a single gene in the Dunaliella genome.     

Anti-Herpes Simplex Virus substances produced by the marine green alga, Dunaliella primolecta

Among 106 microalgae tested, the cytopathic effect (CPE) upon Vero cells of herpes simplex virus, Type 1 (HSV-1) was inhibited by four methanol extracts of Dunaliella bioculata C-523, D. primolecta C-525, Lyngbya sp. M-9 and Lyngbya aerugineo-coerulea M-12. The green alga, D. primolecta, had the highest anti HSV-1 activity, since 10 μg mL-1 of extract from this alga completely inhibited the CPE. This activity was similar to that of acyclovir at the same concentration. We compared anti-viral activities against adeno virus, herpes simplex virus-2 (HSV-2), Japanese Encephalitis and Polio viruses. Only the CPE of HSV-2 was inhibited. Thus, the factor was specific against HSV. The antiviral activity was apparently excited during HSV adsorption and invasion of the cells. We optimized the conditions for anti HSV-1 activity by prolonging the exposure of HSV-1 to the extract. After 2 h, the CPE of even a high titer of HSV-1 (106 TCID50/0.1 mL) was completely inactivated. By use of various chromatographic techniques, three green substances having anti-HSV activity were purified from the algal mass of D. primolecta, and 5 μg mL-1 of this purified substances completely inhibited the CPE. From the analysis of NMR and MS, the chemical structures of the active substances were identified as pheophorbide-like compounds. This revised version was published online in June 2006 with corrections to the Cover Date.     

Low-CO2-inducible protein synthesis in the green alga Dunaliella tertiolecta

In the green marine alga Dunaliella tertiolecta, a CO₂-concentrating mechanism is induced when the cells are grown under low-CO₂ conditions (0.03% CO₂). To identify proteins induced under low-CO₂ conditions the cells were labelled with ³⁵SO₄ ^2–, and seven polypeptides with molecular weights of 45, 47, 49, 55, 60, 68 and 100 kDa were detected. The induction of these polypeptides was observed when cells grown in high CO₂ (5% CO₂ in air) were switched to low CO₂, but only while the cultures were growing in light. Immunoblot analysis of total cell protein against pea chloroplastic carbonic anhydrase polyclonal antibodies showed immunoreactive 30-kDa bands in both high- and low-CO₂-grown cells and an aditional 49-kDa band exclusively in low-CO₂-grown cells. The 30-kDa protein was shown to be located in the chloroplast. Western blot analysis of the plasmamembrane fraction against corn plasma-membrane AT-Pase polyclonal antibodies showed 60-kDa bands in both high- and low-CO₂ cell types as well as an immunoreactive 100-kDa band occurring only in low-CO₂-grown cells. These results suggest that there are two distinct forms of both carbonic anhydrase and plasma-membrane ATPase, and that one form of each of them can be regulated by the CO₂ concentration.Abbreviations CA carbonic anhydrase - DIC dissolved inorganic carbon (CO₂+ HCO₃ ^–) - CCM CO₂-concentrating mechanism - low CO₂ air containing 0.03% CO₂ - high CO₂ air supplemented with 5% CO₂ (v/v) We thank Prof. John Coleman for providing antibodies raised against pea chloroplast CA, Dr. James V. Moroney for providing antibodies raised against the 37-kDa periplasmic carbonic anhydrase of CO₂ Chlamydomonas reinhardtii, and Prof. Leonard T. Robert for a gift of corn plasma-membrane 100-kDa ATPase antibodies. We thank Dr. Jeanine Olsen (University of Groningen, the Netherlands) for style comments. This work was supported by the Institute Tecnológico de Canarias (Spain).     

Preparation of intact chloroplasts by chemically induced lysis of the green alga Dunaliella marina

A method for the isolation in high yield of intact chloroplasts from the unicellular green alga Dunaliella marina (Volvocales) is described. This procedure uses chemically induced lysis of cells with the polycationic macromolecules, DEAE-dextran (M=500,000) or poly-D,l-lysine (M=30,000-70,000). Reaction conditions were optimized with respect to obtaining a high yield of intact chloroplasts, after isopycnic centrifugation in a linear sucrose density gradient, by varying the concentration of polycation and the temperature and pH of incubation. Broken chloroplasts devoid of the stromal marker enzymes fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, but containing mitochondrial (fumarase) and microbody (catalase) contamination, were banded at a bouyant density of 1.18 g cm^-3. Intact chloroplasts, as indicated by their retention of alkaline fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, were found in 30% yield (chlorophyll in intact cells, 100%) at an equilibrium density of 1.24 g cm^-3. Contamination by cytoplasmic material (pyruvate kinase), mitochondria, and microbodies was less than 8% each.Abbreviations Chl chlorophyll - DEAE-dextran diethylaminoethyl-dextran - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - FBPase fructose-1,6-bisphosphate phosphatase, EC 3.1.3.11 - G6P-DH glucose 6-phosphate dehydrogenase, EC 1.1.1.49 - HEPES N-2-hydroxyethylpiperazine-N-ethanesulphonic acid - MES 2-(N-morpholino)ethanesulphonic acid - RuBP carboxylase D-ribulose-1,5-bisphosphate carboxylase or 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39     

Photobleaching in the unicellular green alga Dunaliella parva 19/9

The change in the pigment composition of the unicellular alga Dunaliella parva 19/9 during exposure to high light (4000 mol m^-2 s^-1) has been investigated. During photobleaching the carotenoids were lost at a greater rate than the chlorophylls. In these photoinhibitory conditions, -carotene and especially the minor carotenes, - and -carotene, were more susceptible to oxidative destriction than the xanthophylls. Lutein, the major carotenoid present, was the most stable of the carotenoids in these conditions. In addition to the direct photobleaching of carotenoids and chlorophylls, high light treatment induced the de-epoxidation of violaxanthin to antheraxantin and zeaxanthin. Small amounts of zeaxanthin were present in cells prior to illumination but the amount increased 2.4 fold following high light treatment. The effects of extremes of temperature during exposure to high light intensities were also investigated. The destruction of chlorophylls was found to be more temperature sensitive than that of the carotenoids. The general pattern of loss for the individual carotenoids was similar to that found at 25°C, i.e., the carotenes were more readily degraded than the xanthophylls.     

Production of phytoene by herbicide-treated microalgae Dunaliella bardawil in two-phase systems

In the present study we have optimized the concentration of the bleaching herbicide norflurazon to obtain Dunaliella bardawil cells able to accumulate phytoene without losing viability. The highest concentration of phytoene 10.4 g/gChl was obtained for a concentration of norflurazon of 10 microg/mL. Norflurazon-treated Dunaliella bardawil cells are able to accumulate high concentrations of phytoene if the carotenogenic pathway is stimulated, but the lack of colored carotenoids make these cells particularly sensitive to high light intensities and to UVB radiation, so other stimuli, such as nitrogen starvation, have to be used to force the accumulation of phytoene. Detailed time-course evolution of the carotenoids lutein, violaxanthin, zeaxanthin, phytene and beta-carotene and the photosynthetic pigment chlorophyll was followed upon transfer of Dunaliella bardawil cells to nitrogen starvation in presence and absence of norflurazon. The combined use of the carotenogenic pathway inhibitor norflurazon and biphasic aqueous/organic systems to force the excretion of phytoene into the culture medium has been investigated. Cells cultured in the biphasic system were viable and able to produce phytoene during 3 days. Futhermore the productivity increased from 0.14 g/gChl . h in the aqueous culture to 0.18 g/gChl . h in the biphasic system. About 15% of the total phytoene produced by Dunaliella bardawil was excreted and immediately partionated into the organic phase. The concentration of phytoene in the decane phase was 2.05 g/gChl after 72 h, this means that about 47 g of phytoene per litre of culture were in the organic phase.     

Stereoisomers of beta-Carotene and Phytoene in the Alga Dunaliella bardawil

Dunaliella bardawil, a halotolerant green alga, was previously shown to accumulate high concentrations of β-carotene when grown outdoors under defined conditions. The β-carotene of algae cultivated under high light intensity in media containing a high salt concentration is composed of approximately 50% all-trans β-carotene and 40% 9-cis β-carotene. We show here that the 9-cis to all-trans ratio is proportional to the integral light intensity to which the algae are exposed during a division cycle. In cells grown under a continuous white light of 2000 microeinsteins per square meter per second, the ratio reached a value of around 1.5, while in cells grown under a light intensity of 50 microeinsteins per square meter per second, the ratio was around 0.2. As previously shown, algae treated with the herbicide norflurazon accumulate phytoene in place of β-carotene. Electron micrographs showed that the phytoene is accumulated in many distinct globules located in the interthylakoid spaces of the chloroplast. Here too, two isomers are present, apparently all-trans and 9-cis phytoene, and their ratio is dependent upon the integral light intensity to which the algae are exposed during a division cycle. In the presence of norflurazon, Dunaliella bardawil grown under a light intensity of 2000 microeinsteins per square meter per second contained about 8% phytoene with a 9-cis to all-trans ratio of about 1.0. This ratio decreased to about 0.1 when the light intensity was reduced to 50 microeinsteins per square meter per second. These data suggest that the isomerization reaction which leads to the production of the 9-cis isomer occurs early in the path of carotene biosynthesis, at or before the formation of all-trans phytoene. The presence of the 9-cis isomer of β-carotene and the dependence of its preponderance on light intensity seem to be a common feature of many plant parts. Thus carrots which are exposed to minimal light contain no 9-cis isomer while sun-exposed leaves, fruits, and flowers contain 20 to 50% of the 9-cis isomer.     

Regulation of phytochelatin synthesis by zinc and cadmium in marine green alga,Dunaliella tertiolecta

Although Cd(2+) is a more effective inducer of phytochelatin (PC) synthesis than Zn(2+) in higher plants, we have observed greater induction of PC synthesis by Zn(2+) than Cd(2+) in the marine green alga, Dunaliella tertiolecta. To elucidate this unique regulation of PC synthesis by Zn(2+), we investigated the effects of Zn(2+) and Cd(2+) on the activities of both phytochelatin synthase (PC synthase) and enzymes in the GSH biosynthetic pathway. PC synthase was more strongly activated by Cd(2+) than by Zn(2+), but the difference was not very big. On the other hand, gamma-glutamylcysteine synthetase (gamma-ECS) and glutathione synthetase (GS) were activated by both heavy metals, but their activities were higher in Zn-treated cells than in Cd-treated cells. Dose-dependent stimulation of intracellular reactive oxygen species (ROS) production was observed with Zn(2+), but not Cd(2+) treatment. These results suggest that Zn(2+) strongly promotes the synthesis of GSH through indirect activation of gamma-ECS and GS by stimulating ROS generation. This acceleration of the flux rate for GSH synthesis might mainly contribute to high level PC synthesis.     

Effects of salinity increase on carotenoid accumulation in the green alga Dunaliella salina

The effect of sudden salinity increases on the kinetics of growth and carotenogenesis was studied in three geographically diverse isolates of Dunaliella saliva. A sudden increase in salinity results in a lag phase in growth and the length of this lag phase is dependent on the final salinity and the magnitude of the salinity change (no lag at 10–15% w/v NaCl, 4-day lag at 30% NaCl). There is also a lag before an increase in the total carotenoid content can be measured following the salinity up-shock, and the length of the lag depends largely on the initial salinity and the magnitude of the salinity up-shock, whereas the rate of carotenogenesis and the final carotenoid content reached depend on the final salinity. The increase in total carotenoid content is mainly due to β-carotene. Following the salinity up-shock (especially from 10% to 20% NaCl) the proportion of lutein as a percentage of total carotenoids decreases, whereas zeaxanthin increases. This suggests that the pathway synthesising lutein is more sensitive to salt or osmotic stress and is inhibited at higher salinities, thus leading to β-carotene formation. The proportion of α-carotene does not change.     

Identification of a mitochondrial nucleoside diphosphate kinase from the green alga Dunaliella tertiolecta

We isolated a full-length cDNA encoding a nucleoside diphosphate (NDP) kinase from a Dunaliella tertiolecta cDNA library by homology cloning and rapid amplification of cDNA ends-PCR. The cDNA sequence, consisting of 840 bp, contained an open reading frame coding for a 221-amino acid protein. The predicted 24-kDa protein was named DtNDK1. It possesses all the residues involved in nucleotide binding and catalysis and, in its long N-terminus, contains putative mitochondrial targeting peptides. The full-length pre-protein expressed in Escherichia coli as a recombinant N-terminally His-tagged protein was retained in inclusion bodies, totally devoid of NDP kinase activity. Upon expression in yeast cells, the full-length protein His-tagged at the C-terminus was found processed in a soluble form that was lacking the first 67 amino acids from the N-terminus. The mature protein, which was purified by affinity chromatography to near homogeneity, showed NDP kinase activity. Confocal microscopy on yeast cells expressing the recombinant protein revealed the specific mitochondrial localization of DtNDK1 labeled at the C-terminus with green fluorescent protein.     

The salt relations of marine and halophilic species of the unicellular green alga,Dunaliella

1. Comparisons were made of the effects of salt on the exponential growth rates of two unicellular algae,Dunaliella tertiolecta (marine) andDunaliella viridis (halophilic).
2. The algae contained glycerol in amounts which varied directly with the salt concentration of the growth media. The highest measured glycerol content ofD. tertiolecta was approximately equivalent to 1.4 molal and occurred in algae grown in 1.36 M sodium chloride. The highest glycerol content measured inD. viridis was approximately equivalent to 4.4 molal and occurred in algae grown in 4.25 M sodium chloride. Lower concentrations of free glucose, which varied inversely with extracellular salt concentration, were also detected.
3. It is inferred that Na⁺ is effectively excluded from the two algae. There was some evidence of a moderate uptake of K⁺.
4. Comparisons were made of erude preparations of the glucose-6-phosphate dehydrogenase and an NADP-specific glycerol dehydrogenase from each species and of the effects of salt and glycerol on the activities of these enzymes. It is concluded that the different salt tolerances of the two algae cannot be explained by generalized differences between their enzyme proteins.
5. Although intracellular glycerol must necessarily contribute to the osmotic status of the algae, its primary function in influencing their salt relations is considered to be that of a compatible solute, whereby glycerol maintains enzyme activity under conditions of high extracellular salt concentration and hence low (thermodynamic) water activity.

     

The respiratory inhibitor antimycin A specifically binds Fe(III) ions and mediates utilization of iron by the halotolerant alga Dunaliella salina (Chlorophyta)

It is demonstrated that Antimycin A (AA), a respiratory inhibitor produced by Streptomyces bacteria, forms lipophylic complexes with Fe(III) ions. Spectroscopic titration indicates that Fe(III) ions interact with 2AA molecules. At growth-limiting Fe concentrations, AA mediates Fe uptake and promotes growth and chlorophyll synthesis better than other Fe chelators in the halotolerant alga Dunaliella salina. It is proposed that AA enhances Fe bioavailability in hypersaline solutions by formation of lipophylic Fe-AA complexes which are taken-up and utilized by the algae. The results suggest that the respiratory inhibitor AA can affect Fe metabolism in microorganisms.     

Photoinduction of Massive beta-Carotene Accumulation by the Alga Dunaliella bardawil: Kinetics and Dependence on Gene Activation

The massive accumulation of β-carotene by the halotolerant micro alga Dunaliella bardawil, in response to high light intensity and several other environmental factors, has been studied so far under different sets of fixed conditions. To determine the kinetics and characteristics of the induction of β-carotene accumulation, cells continuously grown under white light of approximately 27 microeinsteins per square meter per second were exposed to light of approximately 1650 microeinsteins per square meter per second. The exposed cells accumulate β-carotene in two stages: the first stage, lasting for 24 hours, starts shortly after exposure, whereas the second stage starts concomitantly with the onset of the stationary phase and persists until the cells collapse. Actinomycin D, chloramphenicol, or cycloheximide added to low-illuminated cultures abolish the subsequent induction of β-carotene accumulation by high light intensity. These results, together with the early exponential kinetics of accumulation, point to the role of gene activation in the process. In vivo labeling of proteins and in vitro translation of poly(A)⁺ mRNA revealed several pronounced differences between low-illuminated and high-illuminated cells. A strongly light-induced protein of approximately 55 kilodaltons, as well as other light-induced proteins could possibly fulfill a carotenogenic function.     

Strategies for the allocation of resources under sulfur limitation in the green alga Dunaliella salina

The effect of sulfur limitation on the partitioning of carbon, nitrogen, and sulfur was investigated in Dunaliella salina. D. salina was able to adapt to 6 microM sulfate; under these conditions, the cells showed reduced growth and photosynthetic rates. Whereas intracellular sulfate was depleted, phosphate, nitrate, and ammonium increased. Amino acids showed a general increase, and alanine became the most abundant amino acid. The activities of four key enzymes of carbon, sulfur, and nitrogen metabolism were differentially regulated: Adenosine 5' triphosphate sulfurylase activity increased 4-fold, nitrate reductase and phosphoenolpyruvate (PEP) carboxylase activities decreased 4- and 11-fold, respectively, whereas carbonic anhydrase activity remained unchanged. Sulfur limitation elicited specific increase or decrease of the abundance of several proteins, such us Rubisco, PEP carboxylase, and a light harvesting complex protein. The accumulation of potentially toxic ammonium indicates an insufficient availability of carbon skeletons. Sulfur deficiency thus induces an imbalance between carbon and nitrogen. The dramatic reduction in PEP carboxylase activity suggests that carbon was diverted away from anaplerosis and possibly channeled into C3 metabolism. These results indicate that it is the coordination of key steps and components of carbon, nitrogen, and sulfur metabolism that allows D. salina to adapt to prolonged sulfur limitation.     

A mutant of the green alga Dunaliella salina constitutively accumulates zeaxanthin under all growth conditions

A novel mutant (zea1) of the halotolerant unicellular green alga Dunaliella salina is impaired in the zeaxanthin epoxidation reaction, thereby lacking a number of the beta-branch xanthophylls. HPLC analysis revealed that the zea1 mutant lacks neoxanthin (N), violaxanthin (V) and antheraxanthin (A) but constitutively accumulates zeaxanthin (Z). Under low-light physiological growth conditions, the zea1 (6 mg Z per g dry weight or 8 x 10(-16) mol Z/cell) had a substantially higher Z content than the wild type (0.2 mg Z per g dry weight or 0.5 x 10(-16) mol Z/cell). Lack of N, V, and A did not affect photosynthesis or growth of the zea1 strain. Biochemical analyses suggested that Z constitutively and quantitatively substitutes for N, V, and A in the zea1 strain. This mutant is discussed in terms of its commercial value and potential utilization by the algal biotechnology industry for the production of zeaxanthin, a high-value bioproduct.