Photosynthetic characteristics of Dunaliella salina (Chlorophyceae,Dunaliellales) in relation to β-carotene content

Photosynthetic characteristics of Dunaliella salina with high (red form) and low β-carotene (green form) concentrations were studied. D. salina growing in brine saltworks exhibited a high level of β-carotene (15 pg cell⁻¹). The rate of oxygen evolution as a function of irradiance was higher in the red than in the green form (on chlorophyll basis). Photosynthetic inhibition of the green form was observed above 500 μmol m⁻² s⁻¹. The red form appeared more resistant to high irradiance and no inhibition in O₂ evolution was observed up 2000 μmol m⁻² s⁻¹. However, when these results are expressed on a cell number basis the rate of oxygen evolution was significantly higher in the green form. Carbonic anhydrase (CA) activity (total, soluble, membrane bound) was found in red and green forms. CA was higher in the red form on a chlorophyll basis, but lower if expressed on a protein basis. The light dependent rate of oxygen evolution and photoinhibition depends on the concentration of β-carotene in D. salina cells.     

The biosynthetic pathway of carotenoids in the astaxanthin-producing green alga <Emphasis Type="Italic">Chlorella zofingiensis</Emphasis>

The carotenoid composition of the astaxanthin-producing green alga Chlorella zofingiensis was investigated using high-performance liquid chromatography. Astaxanthin, adonixanthin, and zeaxanthin are the major carotenoids in this alga. The pigment pattern was characterized during the accumulation period, and in response to diphenylamine (DPA), an inhibitor of carotenoid biosynthesis. An increase in zeaxanthin followed by a decrease in xanthophyll was seen after the induction of astaxanthin biosynthesis by glucose. This biphasic kinetics of zeaxanthin was parallel to the marked increase in adonixanthin (from 0 mg g⁻¹ to 0.21 mg g⁻¹) and astaxanthin (from 0.05 mg g⁻¹ to 0.35 mg g⁻¹) and decrease of β-carotene (from 0.30 mg g⁻¹ to 0.03 mg g⁻¹). More importantly, unlike the Haematococcus alga, in which there was a high β-carotene accumulation after DPA treatment, C. zofingiensis showed an accumulation of extra zeaxanthin instead of β-carotene after treatment of the cells with DPA. All these results observed in vivo studies corroborate the observations in vitro studies at the enzyme level that zeaxanthin is a substrate for the carotenoid oxygenase in C. zofingiensis. It is suggested that zeaxanthin might be an important intermediate and not an end product of the biosynthetic pathway of astaxanthin. Therefore, a new pathway for astaxanthin formation by C. zofingiensis, which is different from that of the other astaxanthin-producing microorganisms, is proposed. An understanding of the astaxanthin biosynthetic pathway may yield important information toward the optimization of astaxanthin production, especially for the improvement of astaxanthin through genetic manipulations.     

Astaxanthin biosynthesis enhanced by reactive oxygen species in the green algaHaematococcus pluvialis

The unicellular green algaHaematococcus pluvialis has recently attracted great interest due to its large amounts of ketocarotenoid astaxanthin, 3,3′-dihydroxy-β,β-carotene-4,4′-dione, widely used commercially as a source of pigment for aquaculture. In the life cycle ofH. pluvialis, astaxanthin biosynthesis is associated with a remarkable morphological change from green motile vegetative cells into red immotile cyst cells as the resting stage. In recent years we have studied this morphological process from two aspects: defining conditions governing astaxanthin biosynthesis and questioning the possible function of astaxanthin in protecting algal cells against environmental stress. Astaxanthin accumulation in cysts was induced by a variety of environmental conditions of oxidative stress caused by reactive oxygen species, intense light, drought, high salinity, and high temperature. In the adaptation to stress, abscisic acid induced by reactive oxygen species, would function as a hormone in algal morphogenesis from vegetative to cyst cells. Furthermore, measurements of bothin vitro andin vivo antioxidative activities of astaxanthin clearly demonstrated that tolerance to excessive reactive oxygen species is greater in astaxanthin-rich cysts than in astaxanthin-poor cysts or astaxanthin-less vegetative genesis and carotenogenesis, and the accumulated astaxanthin in cysts can function as a protective agent against oxidative stress damage. In this study, the physiological roles of astaxanthin in stress response and cell protection are reviewed.     

Increases in cell elongation,plastid compartment size and phytoene synthase activity underlie the phenotype of the<Emphasis Type="Italic"> high pigment-1</Emphasis> mutant of tomato

A characteristic trait of the high pigment-1 ( hp-1) mutant phenotype of tomato ( Lycopersicon esculentum Mill.) is increased pigmentation resulting in darker green leaves and a deeper red fruit. In order to determine the basis for changes in pigmentation in this mutant, cellular and plastid development was analysed during leaf and fruit development, as well as the expression of carotenogenic genes and phytoene synthase enzyme activity. The hp-1 mutation dramatically increases the periclinal elongation of leaf palisade mesophyll cells, which results in increased leaf thickness. In addition, in both palisade and spongy mesophyll cells, the total plan area of chloroplasts per cell is increased compared to the wild type. These two perturbations in leaf development are the primary cause of the darker green hp-1 leaf. In the hp-1 tomato fruit, the total chromoplast area per cell in the pericarp cells of the ripe fruit is also increased. In addition, although expression of phytoene synthase and desaturase is not changed in hp-1 compared to the wild type, the activity of phytoene synthase in ripe fruit is 1.9-fold higher, indicating translational or post-translational control of carotenoid gene expression. The increased plastid compartment size in leaf and fruit cells of hp-1 is novel and provides evidence that the normally tightly controlled relationship between cell expansion and the replication and expansion of plastids can be perturbed and thus could be targeted by genetic manipulation.     

Functional analysis of multiple carotenogenic genes from <Emphasis Type="Italic">Lycium barbarum</Emphasis> and <Emphasis Type="Italic">Gentiana lutea</Emphasis> L. for their effects on β-carotene production in transgenic tobacco

Carotenoids are red, yellow and orange pigments, which are widely distributed in nature and are especially abundant in yellow-orange fruits and vegetables and dark green leafy vegetables. Carotenoids are essential for photosynthesis and photoprotection in plant life and also have different beneficial effects in humans and animals (van den Berg et al. 2000). For example, β-carotene plays an essential role as the main dietary source of vitamin A. To obtain further insight into β-carotene biosynthesis in two important economic plant species, Lycium barbarum and Gentiana lutea L., and to investigate and prioritize potential genetic engineering targets in the pathway, the effects of five carotenogenic genes from these two species, encoding proteins including geranylgeranyl diphosphate synthase, phytoene synthase and δ-carotene desaturase gene, lycopene β-cyclase, lycopene ε-cyclase were functionally analyzed in transgenic tobacco (Nicotiana tabacum) plants. All transgenic tobacco plants constitutively expressing these genes showed enhanced β-carotene contents in their leaves and flowers to different extents. The addictive effects of co-ordinate expression of double transgenes have also been investigated.     

Carotenes from Mutants of the Dinoflagellate, Crypthecodinium cohnii

SYNOPSIS.
The carotenoid compositions of 15 nitrosoguanidine-induced mutants of Crypthecodinium cohnii , a heterotrophic dinoflagellate, were determined by chromatographic and mass spectral analyses. Wild-type C. cohnii grown with irradiation of 250 W/cm² visible light at 27 C synthesizes β-carotene (33%) and γ-carotene (67%) amounting to 0.083 mg/g dry wt. There are 4 types of carotenoid-deficient mutants: (I) albinos which synthesize no C₄₀-carotonoids: (II) albinos blocked at the level of phytoene desaturation; (III) cream-colored cells which accumulate mainly §–carotene, with phytoene and/or β-zeacarotene also present; and (IV) light-orange strains which synthesize reduced amounts of β-carotene and γ-carotene.
Dark-grown wild-type cells produced 35% as much carotenoids as light-grown cells. Inhibition studies revealed that diphenylamine (3 γ) caused phytoene accumulation; nicotine at 0.9 mM blocked the final cyclization, to cause γ-carotene to accumulate in wild-type cells. Inhibition by adenine and guanine (1.5 mM) of carotenogenesis was demonstrated for the first time in any system. The effect of these purines was similar to that of diphenylamine addition: phytoene desaturation was largely inhibited.
The carotenogenic system in this dinoflagellate is similar to that of green algae and higher plants, and is under nuclear genetic control.     

Analysis of proteomic changes in colored mutants of Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin as its most prevalent xanthophyll derivative. Comparisons between the protein profiles of mutant lines of this yeast can provide insight into the carotenogenic pathway. Differently colored mutants (red, orange, pink, yellow, and white) were obtained from this yeast species, and their protein profiles were determined using two-dimensional polyacrylamide gel electrophoresis (2DE). Individual proteins differentially expressed were identified using mass spectrometry. The red mutants hyperproduced total carotenoids (mainly astaxanthin), while in white and orange mutants, mutagenesis affected the phytoene dehydrogenase activity as indicated by the accumulation of phytoene. Inactivation of astaxanthin synthase after the mutagenic treatment was evident in β-carotene accumulating mutants. Differences in the proteomic profiles of wild-type X. dendrorhous and its colored mutants were demonstrated using 2DE. Of the total number of spots detected in each gel (297–417), 128 proteins were present in all strains. The red mutant showed the greatest number of matches with respect to the wild type (305 spots), while the white and yellow mutants, which had reduced concentrations of total carotenoids, presented the highest correlation coefficient (0.6) between each other. A number of differentially expressed proteins were sequenced, indicating that tricarboxylic acid cycle and stress response proteins are closely related to the carotenogenic process.     

Molecular evolution of carotenoid biosynthesis from bacteria to plants

β-Carotene and derivatives are important pigments in plant photosynthesis. They are found not only in green plants but also accumulate in archea, prokaryotes and fungi. For β -carotene biosynthesis, enzymes are necessary to catalyse the formation of phytoene, several desaturation steps and cyclization reactions. This review is focused on the molecular phylogeny of the enzymes, the genes involved and their diversity. It outlines how genes and enzymes from prokaryotes and archea were modified to give rise to the corresponding plant constituents. In the cases of phytoene synthase, a direct line of evolution can be drawn. For other carotenogenic enzymes, new genes and enzymes have been acquired at certain stages of evolution. In addition, phytoene desaturases and lycopene cyclases are examples of convergent evolution of different types of enzymes, which are structurally completely unrelated but functionally identical. Finally, several gene duplications led to homologous enzymes with different catalytic functions including those involved in the synthesis of α -carotene.     

Prenyl lipid and fatty-acid synthesis in isolatedAcetabularia chloroplasts

A gentle procedure allowed the isolation of intact and highly active chloroplasts from the unicellular green algaAcetabularia mediterranea. These chloroplasts incorporated carbon from NaH¹⁴CO₃ into fatty acids and prenyl lipids at a rate of about 20–50 nmol carbon· (mg chlorophyll)⁻¹·h⁻¹. Most of the fatty acids formed in vitro were esterified in galactolipids. The main prenyl lipids synthesized were the chlorophyll side chain, intermediates of the carotenogenic path, α-and β-carotene, as well as lutein. Large amounts of [1-¹⁴C]acetate were incorporated, but exclusively into fatty acids.Isopentenyl diphosphate was a good substrate for prenyl-lipid formation in hypotonically treated chloroplasts. The envelope of intact chloroplasts, however, was impermeable to this compound. Intermediates of the mevalonate pathway were not accepted as precursors under conditions whereisopentenyl diphosphate was well incorporated. The results show that the lipid biosynthetic pathways in the plastids ofAcetabularia, a member of the ancient family of Dasycladaceae, are very similar to those in higher-plant plastids. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Genetic basis of microbial carotenogenesis

The synthesis of carotenoids begins with the formation of a phytoene from geranylgeranyl pyrophosphate, a well conserved step in all carotenogenic organisms and catalyzed by a phytoene synthase, an enzyme encoded by the crtB (spy) genes. The next step is the dehydrogenation of the phytoene, which is carried out by phytoene dehydrogenase. In organisms with oxygenic photosynthesis, this enzyme, which accomplishes two dehydrogenations, is encoded by the crtP genes. In organisms that lack oxygenic photosynthesis, dehydrogenation is carried out by an enzyme completely unrelated to the former one, which carries out four dehydrogenations and is encoded by the crtI genes. In organisms with oxygenic photosynthesis, dehydrogenation of the phytoene is accomplished by a ζ-carotene dehydrogenase encoded by the crtQ (zds) genes. In many carotenogenic organisms, the process is completed with the cyclization of lycopene. In organisms exhibiting oxygenic photosynthesis, this step is performed by a lycopene cyclase encoded by the crtL genes. In contrast, anoxygenic photosynthetic and non-photosynthetic organisms use a different lycopene cyclase, encoded by the crtY (lyc) genes. A third and unrelated type of lycopene β-cyclase has been described in certain bacteria and archaea. Fungi differ from the rest of non-photosynthetic organisms in that they have a bifunctional enzyme that displays both phytoene synthase and lycopene cyclase activity. Carotenoids can be modified by oxygen-containing functional groups, thus originating xanthophylls. Only two enzymes are necessary for the conversion of β-carotene into astaxanthin, using several ketocarotenoids as intermediates, in both prokaryotes and eukaryotes. These enzymes are a β-carotene hydroxylase (crtZ genes) and a β-carotene ketolase, encoded by the crtW (bacteria) or bkt (algae) genes. Electronic Publication     

Tissue-specific accumulation of carotenoids in carrot roots

Raman spectroscopy can be used for sensitive detection of carotenoids in living tissue and Raman mapping provides further information about their spatial distribution in the measured plant sample. In this work, the relative content and distribution of the main carrot (Daucus carota L.) root carotenoids, α-, β-carotene, lutein and lycopene were assessed using near-infrared Fourier transform Raman spectroscopy. The pigments were measured simultaneously in situ in root sections without any preliminary sample preparation. The Raman spectra obtained from carrots of different origin and root colour had intensive bands of carotenoids that could be assigned to β-carotene (1,520 cm⁻¹), lycopene (1,510 cm⁻¹) and α-carotene/lutein (1,527 cm⁻¹). The Raman mapping technique revealed detailed information regarding the relative content and distribution of these carotenoids. The level of β-carotene was heterogeneous across root sections of orange, yellow, red and purple roots, and in the secondary phloem increased gradually from periderm towards the core, but declined fast in cells close to the vascular cambium. α-carotene/lutein were deposited in younger cells with a higher rate than β-carotene while lycopene in red carrots accumulated throughout the whole secondary phloem at the same level. The results indicate developmental regulation of carotenoid genes in carrot root and that Raman spectroscopy can supply essential information on carotenogenesis useful for molecular investigations on gene expression and regulation.     

Proteomic analysis of molecular response to oxidative stress by the green alga<Emphasis Type="Italic"> Haematococcus pluvialis</Emphasis> (Chlorophyceae)

Rapidly growing, green motile flagellates of Haematococcus pluvialis can transform into enlarged red resting cysts (aplanospores) under oxidative stress conditions. However, it is not known what initial molecular defense mechanisms occur in response to oxidative stress, and may ultimately lead to cellular transformation. In this study, global-expression profiling of cellular proteins in response to stress was analyzed by two-dimensional gel electrophoresis, image analysis, and peptide mass fingerprinting. Oxidative stress was induced in cultures of green flagellates by addition of acetate and Fe²⁺, and exposure to excess light intensity. Overall, 70 proteins were identified with altered expression patterns following stress induction. Some key proteins involved in photosynthesis and nitrogen assimilation were down-regulated, whereas some mitochondrial respiratory proteins were transiently up-regulated after the onset of stress. Most of the identified proteins, particularly those from the families of superoxide dismutase, catalase, and peroxidase, were transiently up-regulated, but reverted to down-regulation during the 6 days of stress. On the other hand, cellular accumulation of the antioxidant astaxanthin occurred well after initiation of oxidative stress and reached its maximum cellular level after six or more days of stress. It appears that the early stress response involves multiple enzymatic defense processes that play a critical role upon onset of stress and also during the early transition of green vegetative cells to red cysts. As cyst development continues, the intensive, enzyme-mediated initial responses were largely replaced in mature red cysts by accumulation of the molecular antioxidant astaxanthin. This study provides the first direct evidence for a massive, and concerted up-regulation of multiple antioxidative defense mechanisms, both spatially and temporarily, to protect H. pluvialis cells against oxidative stress.Abbreviations 2-DE Two-dimensional gel electrophoresis - IPI Isopentenyl-diphosphate -isomerase - HSP Heat-shock protein - MALDI–TOF Matrix-assisted laser desorption/ionization–time of flight - ROS Reactive oxygen species - SOD Superoxide dismutase     

Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis,and astaxanthin synthesis in Escherichia coli

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes -carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, -carotene ketolase (-carotene oxygenase), which converted -carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3S), which was identified after purification by a variety of spectroscopic methods.     

Three-Dimensional Ultrastructural Study of Oil and Astaxanthin Accumulation during Encystment in the Green Alga Haematococcus pluvialis

Haematococcus pluvialis is a freshwater species of green algae and is well known for its accumulation of the strong antioxidant astaxanthin, which is used in aquaculture, various pharmaceuticals, and cosmetics. High levels of astaxanthin are present in cysts, which rapidly accumulate when the environmental conditions become unfavorable for normal cell growth. It is not understood, however, how accumulation of high levels of astaxanthin, which is soluble in oil, becomes possible during encystment. Here, we performed ultrastructural 3D reconstruction based on over 350 serial sections per cell to visualize the dynamics of astaxanthin accumulation and subcellular changes during the encystment of H. pluvialis. This study showcases the marked changes in subcellular elements, such as chloroplast degeneration, in the transition from green coccoid cells to red cyst cells during encystment. In green coccoid cells, chloroplasts accounted for 41.7% of the total cell volume, whereas the relative volume of astaxanthin was very low (0.2%). In contrast, oil droplets containing astaxanthin predominated in cyst cells (52.2%), in which the total chloroplast volume was markedly decreased (9.7%). Volumetric observations also demonstrated that the relative volumes of the cell wall, starch grains, pyrenoids, mitochondria, the Golgi apparatus, and the nucleus in a cyst cell are smaller than those in green coccid cells. Our data indicated that chloroplasts are degraded, resulting in a net-like morphology, but do not completely disappear, even at the red cyst stage.     

In vivo antioxidant role of astaxanthin under oxidative stress in the green alga Haematococcus pluvialis

Intracellular production of active oxygen in the green alga Haematococcus pluvialis was studied by measuring the capacity for in vivo conversion of 2′,7′-dichlorohydrofluorescein diacetate to the fluorescent dye dichlorofluorescein in different algal cell types (i.e., vegetative, immature cyst and mature cyst cells). The increase in formation of dichlorofluorescein by methyl viologen (superoxide anion radical generator) was linear for 2 h in immature cyst cells (low astaxanthin) in a methyl viologen-concentration-dependent manner, while no production was detected in mature (high astaxanthin) cysts. Compared to cyst cells, formation of dichlorofluorescein in vegetative cells (no astaxanthin) was markedly increased by methyl viologen. The formation of dichlorofluorescein in cyst cells was decreased with higher astaxanthin content under excessive oxidative stress. All of the active oxygen species tested (singlet oxygen, superoxide anion radical, hydrogen peroxide and peroxy radical) at 10⁻³ M increased the intracellular dichlorofluorescein formation in immature cysts, but did not increase the dichlorofluorescein content of mature cysts. Therefore, astaxanthin in cyst cells appeared to function as an antioxidant agent against oxidative stress. Received: 26 January 2000 / Received revision: 5 April 2000 / Accepted: 1 May 2000     

Dynamic Changes of Photosynthetic Pigments in Soybean Callus under High Irradiance

Dynamic changes of neoxanthin (NEO), violaxanthin (VIO), anteraxanthin (ANT), zeaxanthin (ZEA), chlorophyll (Chl) a, Chl b, α-carotene, β-carotene, and their behaviour under increasing duration of high irradiance (HI) were investigated in the soybean hypocotyl callus culture. The calli were induced on solid (1.1 % agar) MS medium (pH 5.8) supplemented with 4.52 μM 2,4-D, 2.32 μM kinetin, and 3 % sucrose. After 30 d of culture, the green calli were irradiated with “white light” (133W m⁻²) for 0, 3.5, and 24 h. HPLC profiles were separated on a C₁₈ column. With increasing duration of HI, the content of total carotenoids (Cars) increased, but the ratio of Chl a+b/Cars decreased. With lengthening the duration of HI, there was induction of ZEA. Contents of ANT, α-carotene, and β-carotene remained nearly constant, but ratio of ZEA/Chl a+b increased with lengthening the HI duration. This revised version was published online in August 2006 with corrections to the Cover Date.     

Growth-associated biosynthesis of astaxanthin in heterotrophic Chlorella zofingiensis (Chlorophyta)

The green microalga Chlorella zofingiensis can produce the ketocarotenoid astaxanthin under heterotrophic culture conditions. Here we report the growth-associated biosynthesis of astaxanthin in this biotechnologically important alga. With glucose as sole carbon and energy source, C. zofinginesis grew fast in the dark with rapid exhaustion of nitrogen and carbon sources from media, leading to a high specific growth rate (0.034 h⁻¹). Cultures started at a cell concentration of about 3.4 × 10⁹ cells l⁻¹ reached, after 6 days, standing biomass values of 1.6 × 10¹¹ cells or 8.5 g dry weight l⁻¹. Surprisingly, the biosynthesis of astaxanthin was found to start at early exponential phase, independent of cessation of cell division. A general trend was observed that the culture conditions benefiting cell growth also benefited astaxanthin accumulation, indicating that astaxanthin was a growth-associated product in this alga. The maximum cell dry biomass and astaxanthin yield were 11.75 g l⁻¹ and 11.14 mg l⁻¹ (about 1 mg g⁻¹), simultaneously obtained in the fed-batch culture with a combined glucose–nitrate mixture addition, which were the highest ever reported in dark-heterotrophic algal cultures. The possible reasons why dark-heterotrophic C. zofingiensis could produce astaxanthin during the course of cell growth were discussed.