L'infrasTruttura di Dunaliella Salina

Abstract

The ultrastructure of Dunaliella salina. — The ultrastructure of Dunaliella salina is described. Its structural organisation is rather similar to that previously reported for other Volvocales. The lack of a cellulosic wall is confirmed; the protoplast is bounded only by the monolayered plasma membrane which covers also the flagella. The conspicuous cup-shaped chloroplast fills up the whole back part of the cell and its terminal part is close to the anterior pole: consequently all the other cellular organelles are confined in its deep anterior cavity. The pyrenoid is located in the back part of the chloroplast: communication between the pyrenoid and the surrounding chloroplast is established by a system of paired lamellae. The droplets of carotenoids which form the eyespot are not stacked in regular order. The Golgi apparatus is located in the anterior part and is formed by two conspicuous dictyosomes. A great number of vesciculae, perhaps formed by the dictyosome, are crowded around the insertion of the two flagella. Evidence for the existence of an endoplasmic reticulum is uncertain. Two unknown structures are observed and their morphological organisation is described.     

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.     

新型生物反应器——杜氏盐藻研究进展

转基因植物作为生物反应器生产外源物质已成为基因工程领域的研究热点之一 ,而杜氏盐藻 (Dunaliellasalina)作为新型生物反应器生产外源蛋白具有独特的优点 ,就盐藻这一生物反应器的特点、存在问题和开发应用前景等的最近研究进展作一简要综述。     

Plasma membrane potential of the alga dunaliella, and its relation to osmoregulation

A fluorescent dye sensitive to membrane potential was used to follow the plasma-membrane potential in the unicellular halo-tolerant alga Dunaliella salina. The signal observed during dissipation of the plasma membrane potential by the addition of excess K⁺ and valinomycin, or a protonophore, was taken as a measure of the preexisting potential. A resting potential of −85 to −100 millivolts (negative inside) was calculated. Following a hypertonic shock, the plasma membrane was rapidly hyperpolarized. This hyperpolarization was transient, and the algae resumed their resting potential about 30 minutes after the shock. The resting plasma membrane potential was decreased by vanadate and is concluded to be generated mostly by the plasma membrane ATPase of Dunaliella. The transient hyperpolarization following a hypertonic shock indicates, therefore, a transient activation of the ATPase. This is further corroborated by a rapid transient decrease in the intracellular ATP following a hypertonic shock and its inhibition by vanadate. It is suggested that activation of the plasma membrane ATPase may be the trigger for osmoregulation in Dunaliella.     

ANALYSIS OF THE EXPRESSION OF GENES FOR P-TYPE ATPASE IN THE HALOTOLERANT ALGA DUNALIELLA SALINA (CHLOROPHYCEAE)1

A partial complementary DNA (cDNA) (DSA8) for a P-type ATPase was obtained from the halotolerant alga Dunaliella salina (Dunal) Teod. (Chlorophyceae). The cDNA exhibited greater than 90% homology to the cDNA for a H⁺-ATPase in D. bioculata Butcher. The expression of the gene that corresponded to DSA8 was decreased strongly by increases in NaCl concentration. The expression of a gene that corresponded to another ATPase (DSA1; possibly for a Ca²⁺-ATPase) from D. salina did not show the same decrease as did the DSA8. However, increased osmotic pressure due to glycerol resulted in the same decrease in the DSA8 gene. Under salt or osmotic stress, the activity of a H⁺-ATPase from microsomes of this alga also decreased. We suggest that expression of the gene for the plasma membrane H⁺-ATPase of D. salina is regulated by osmotic pressure rather than by the concentration of NaCl.     

盐藻蛋白质组的研究进展

盐藻是一种公认的能耐受高盐度的海藻,是研究植物高盐适应的模式生物.高通量的蛋白质组学为人们深入探讨盐藻的耐盐机制提供了强有力的工具.就蛋白质组及其主要技术、盐藻蛋白质组的研究概况和研究展望作一简要综述.     

Dunaliella salina: A model System for Studying the Response of Plant Cells to Stress

This review focuses on the biochemical and physiological responseof the halotolerant green alga, Dunaliella salina, to conditionsof stress. It is now well established that in response to stress,cells of Dunaliella salina var. bardawil show increased glycerolproduction, massive ßcarotene accumulation and enhancedabscisic acid metabolism. In this respect, cellular responsesare regulatory and seem to depend on a diversity of mechanismswhich may be linked to a modification of the abscisic acid balance.Dunaliella lacks a rigid cell wall and the cellular contentsare enclosed by an elastic plasma membrane that permits rapidcell volume changes in response to extracellular changes inosmolarity. Based on the ‘stretch activated ion channelsmodel’ reviewed recently by Kirst (1990) we propose thefollowing cascade of responses: volume change/distortion ofplasmalemma     

杜氏盐藻分子生物学最新进展及展望

杜氏盐藻是一种无细胞壁的单细胞双鞭毛真核藻类,是一种十分重要的藻类资源。过去对杜氏盐藻的研究多集中在形态学、耐盐机理及β-胡萝卜素等方面,近年来,随着藻类基因工程的快速发展,本研究课题组及国内外在杜藻盐藻分子生物学方面做了大量工作,现就杜氏盐藻在这一领域的研究进展进行综述,主要是重要功能基因的克隆与分析、杜氏盐藻调控序列的研究以及杜氏盐藻作为宿主表达外源基因等。     

Hydrolysis of polyphosphates and permeability changes in response to osmotic shocks in cells of the halotolerant alga dunaliella

The effects of osmotic shocks on polyphosphates and on the vacuolar fluorescent indicator atebrin have been investigated to test whether acidic vacuoles in the halotolerant alga Dunaliella salina have a role in osmoregulation. Upshocks and downshocks induce different patterns of polyphosphate hydrolysis. Upshocks induce rapid formation of new components, tentatively identified as 5 or 6 linear polyphosphates, formed only after upshocks with NaCl and not with glycerol, indicative of compartmentation of Na⁺ into the vacuoles. Conversely, downshocks induce a slower transient accumulation of tripolyphosphates, indicating activation of a different hydrolytic process within the vacuoles. Osmotic shocks do not lead to release of atebrin from acidic vacuoles, indicating that they do not induce a major intravacuolar alkalinization. However, osmotic shocks induce transient permeability changes measured by amine-induced atebrin release from vacuoles. Hypoosmotic shocks transiently increase the permeability (up to 20-fold), whereas hyperosmotic shocks induce a rapid drop in permeability. Electron micrographs of osmotically shocked cells also reveal transient changes in the surface and internal organelles of D. salina cells. It is suggested that hyperosmotic and hypoosmotic shocks induce different changes within acidic vacuoles and in the organization and/or composition of the plasma membrane in Dunaliella.     

Localization of glycolate dehydrogenase in two species of Dunaliella

The occurrence of glycolate oxidase in addition to glycolate dehydrogenase in Dunaliella salina and D. primolecta, as reported in the literature, could not be confirmed. Both species were demonstrated to possess only glycolate dehydrogenase. After separation of organelles by gradient centrifugation, glycolate dehydrogenase along with hydroxypyruvate reductase was found exclusively in the mitochondria. Thus the peroxisomes from Dunaliella are not of the leaf-type: because of their content of catalase, uricase and hydroxyacyl-CoA dehydrogenase they appear to be of the same type as in Eremosphaera and other chlorophycean algae. No activity of glycolate dehydrogenase was found in the chloroplast fraction when the 2,6-dichlorophenol-indophenol test was used.This work was supported by the Deutsche Forschungsgemeinschaft.     

Occurrence of jasmonic acid in Dunaliella (Dunaliellales, Chlorophyta)

The occurrence of jasmonic acid and related compounds in Dunaliella species was investigated using gas-liquid chromatography/mass spectroscopy (GCY MS). Jasmonic acid was identified in the ethyl acetate soluble-acidic fraction of Dunaliella tertiolecta and Dunaliella salina (Dunal) Teodoresco, The concentration of jasmonic acid in D. salina. which is extremely halophilic, was much higher than that in D. tertiolecta Butcher, These results indicate that jasmonic acid might play an important role in salt-tolerance in Dunaliella.     

Effect of Ca2+ channel block on glycerol metabolism in Dunaliella salina under hypoosmotic and hyperosmotic stresses

The effect of Ca(2+) channel blockers on cytosolic Ca(2+) levels and the role of Ca(2+) in glycerol metabolism of Dunaliella salina under hypoosmotic or hyperosmotic stress were investigated using the confocal laser scanning microscope (CLSM). Results showed that intracellular Ca(2+) concentration increased rapidly when extracellular salinity suddenly decreased or increased, but the increase could be inhibited by pretreatment of Ca(2+) channel blockers LaCl(3), verapamil or ruthenium red. The changes of glycerol content and G3pdh activity in D. salina to respect to hypoosmotic or hyperosmotic stress were also inhibited in different degrees by pretreatment of Ca(2+) channel blockers, indicating that the influx of Ca(2+) via Ca(2+) channels are required for the transduction of osmotic signal to regulate osmotic responses of D. salina to the changes of salinity. Differences of the three blockers in block effect suggested that they may act on different channels or had different action sites, including influx of Ca(2+) from the extracellular space via Ca(2+) channels localized in the plasma membrane or from intracellular calcium store via the mitochondrial. Other Ca(2+)-mediated or non-Ca(2+)-mediated osmotic signal pathway may exist in Dunaliella in response to hypoosmotic and hyperosmotic stresses.     

A comparative study of sodium andosmotic requirements for growth and nutrient uptake of two related green flagellates,Dunaliella tertiolecta and Chlamydomonas pulsatilla

Dunaliella tertiolecta, a euryhaline marine flagel-late grows over a wide salinity range and exhibits both a minimum sodium and osmotic requirement for growth. Chlamydomonas pulsatilla, a flagellate isolated from a supralittoral rockpool, grows over a more narrow salinity range, and exhibits no sodium or minimum osmotic requirement for growth. The uptake of methylamine by C. pulsatilla is not markedly influenced by the presence of sodium. Phosphate uptake by this flagellate, while showing no absolute dependence on the presence of sodium, is strongly stimulated by low sodium concentrations. Dunaliella tertiolecta, on the other hand, shows absolute requirements for sodium for both methylamine and phosphate uptake. The concentrations of sodium needed for nutrient uptake agree well with those needed for growth when the osmotic requirement for growth of this flagellate is satisfied by mannitol.Abbreviations ASW artificial sea water - MA methylamine     

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

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

The physical state of osmoregulatory solutes in unicellular algae. A natural-abundance carbon-13 nuclear-magnetic-resonance relaxation study.

Natural-abundance 13C n.m.r. spin-lattice relaxation-time measurements have been carried out on intact cells of the unicellular blue--green alga Synechococcus sp. and the unicellular green alga Dunaliella salina, with the aim of characterizing the environments of the organic osmoregulatory solutes in these salt-tolerant organisms. In Synechococcus sp., all of the major organic osmoregulatory solute, 2-O-alpha-D-glucopyranosylglycerol, is visible in spectra of intact cells. Its rotational motion in the cell is slower by a factor of approx. 2.4 than in aqueous solution, but the molecule is still freely mobile and therefore able to contribute to the osmotic balance. In D. salina, only about 60% of the osmoregulatory solute glycerol is visible in spectra of intact cells. The rotational mobility of this observable fraction is approximately half that found in aqueous solution, but the data also indicate that there is a significant concentration of some paramagnetic species in D. salina which contributes to the overall spin-lattice relaxation of the glycerol carbon atoms. The non-observable fraction, which must correspond to glycerol molecules that have very broad 13C resonances and that are in slow exchange with bulk glycerol, has not been properly characterized as yet, but may represent glycerol in the chloroplast. The implications of these findings in relation to the physical state of the cytoplasm and the mechanism of osmoregulation in these cells are discussed.     

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

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

A SALT-INDUCED CHLOROPLAST PROTEIN IN DUNALIELLA SALINA (CHLOROPHYTA)1

The unicellular green alga Dunaliella salina Teod, is halophilic and wall-less. The cell acclimates to osmotic stresses by accumulation or degradation of glycerol. To investigate other mechanisms involved in its physiological recovery following hyperosmotic shocks, protein profiles from cells grown in various salinities were compared. A 13-kDa protein (P13) accumulated when cells were subjected to drastic hyperosmotic shock. Front our results with antibiotic-treated cells and purified chloroplasts, we believe that this component results from de novo translation in chloroplasts. The solubility of P13 was strongly promoted by Triton X-100. Its accumulation was correlated with the recovery of photosynthesis.     

Gene library of Dunaliella salina, prepared in a cosmid vector of new design]

The gene library (56,160 clones) of the halotolerant alga Dunaliella salina was obtained using a novel cosmid vector pBbv-cosII. The mean size of the insertions is 35-45 kb. Hybridization screening demonstrated that both nuclear and chloroplast DNA-containing clones are presented in the library.     

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.Abbreviations PSI(II) photosystem I(II)     

MASSIVE ACCUMULATION OF PHYTOENE INDUCED BY NORFLURAZON IN DUNALIELLA BARDAWIL (CHLOROPHYCEAE) PREVENTS RECOVERY FROM PHOTOINHIBITION1

The effects of nanomolar to micromolar concentrations of the herbicide norflurazon were studied in Dunaliella bardawil Ben-Amotz et Avron, a β-carotene-accumulating halotolerant alga. The large amount of β-carotene which Dunaliella bardawil can contain, around 8% of the algal dry weight, is reduced to 0.2% by treatment with 100 nm norflurazon. Simultaneously, phytoene is accumulated to a similar level of about 8%. The gradual increase in phytoene content, in response to increasing norflurazon concentrations, corresponds to the decrease in β-carotene, with no evident change in other isoprenoid intermediates. Carotene-rich Dunaliella bardawil is substantially resistant to high-intensity photoinhibition. This resistance is lost in cells grown to contain low β-carotene and in the norflurazon-treated phytoene-rich cells. These observations are in agreement with the hypothesis that the accumulated β-carotene in Dunaliella bardawil protects the cells against injury by excessive irradiation.