Some factors governing encystment in Haematococcus pluvialis

Zusammenfassung Die Ergebnisse zeigen, daß die Dauersporenbildung bei dem Flagellaten Haematococcus pluvialis in Kultur durch Störung der Vermehrung bewirkt wird, falls diese nicht eine Folge mangelnder Kohlenstoffassimilation ist. Es hat sich erwiesen, daß anhaltende Assimilation für die Bildung der Dauersporen notwendig ist, ja, daß der Kohlenstoffbedarf dabei größer ist als für bloße Vermehrung.     

Expression of fatty acid synthesis genes and fatty acid accumulation in haematococcus pluvialis under different stressors

Background

Biofuel has been the focus of intensive global research over the past few years. The development of 4^th generation biofuel production (algae-to-biofuels) based on metabolic engineering of algae is still in its infancy, one of the main barriers is our lacking of understanding of microalgal growth, metabolism and biofuel production. Although fatty acid (FA) biosynthesis pathway genes have been all cloned and biosynthesis pathway was built up in some higher plants, the molecular mechanism for its regulation in microalgae is far away from elucidation.

Results

We cloned main key genes for FA biosynthesis in Haematococcus pluvialis, a green microalga as a potential biodiesel feedstock, and investigated the correlations between their expression alternation and FA composition and content detected by GC-MS under different stress treatments, such as nitrogen depletion, salinity, high or low temperature. Our results showed that high temperature, high salinity, and nitrogen depletion treatments played significant roles in promoting microalgal FA synthesis, while FA qualities were not changed much. Correlation analysis showed that acyl carrier protein (ACP), 3-ketoacyl-ACP-synthase (KAS), and acyl-ACP thioesterase (FATA) gene expression had significant correlations with monounsaturated FA (MUFA) synthesis and polyunsaturated FA (PUFA) synthesis.

Conclusions

We proposed that ACP, KAS, and FATA in H. pluvialis may play an important role in FA synthesis and may be rate limiting genes, which probably could be modified for the further study of metabolic engineering to improve microalgal biofuel quality and production.     

Antioxidant role of astaxanthin in the green alga Haematococcus pluvialis

The green unicellular alga, Haematococcus pluvialis has two antioxidative mechanisms against environmental oxidative stress: antioxidative enzymes in vegetative cells and the antioxidative ketocarotenoid, astaxanthin, in cyst cells. We added a reagent that generates superoxide anion radicals (O₂ ⁻), methyl viologen, to mature and immature cysts of H. pluvialis. Tolerance to methyl viologen was higher in mature than in immature cysts. Mature (astaxanthin-rich) cysts showed high antioxidant activity against O₂ ⁻ in permeabilized cells, but not in astaxanthin-free cell extracts, while immature (astaxanthin-poor) cysts had very low antioxidant activities against O₂ ⁻ in both. The results suggested that astaxanthin accumulated in the cyst cells functions as an antioxidant against excessive oxidative stress. The same levels of antioxidant activities against O₂ ⁻ in both permeabilized cells and cell extracts from vegetative cells suggested the presence of antioxidative enzymes (superoxide dismutase). Received: 13 January 1997 / Received revision: 26 February 1997 / Accepted: 27 March 1997     

Abscisic acid-dependent algal morphogenesis in the unicellular green alga Haematococcus pluvialis

To study the physiological role of abscisic acid (ABA) in the unicellular green alga Haematococcus pluvialis, we investigated the effect of ABA on both algal morphogenesis and carotenogenesis in liquid and plate cultures. When ABA was added to vegetative cells of H. pluvialis, red mature cyst cells with enhanced carotenogenesis rapidly appeared on agar plates in Petri dishes. We considered these conditions as drought stress. In plate culture, the morphological change from vegetative to cyst cells was prevented by the inhibitor of chloroplastic protein synthesis, chloramphenicol (CP), resulting in algal death. Exogenous ABA caused recovery of algal encystment even in the presence of CP. The relationship between ABA concentration and morphogenesis in H. pluvialis showed that a decrease in ABA coincided with cyst formation. In contrast, immature cyst cells underwent maturation accompanied by enhanced carotenogenesis in either the presence of CP or the absence of ABA. Therefore, ABA might regulate algal morphogenesis from vegetative to cyst cells, but not carotenogenesis in cyst cells of H. pluvialis. Furthermore, endogenous active oxygen species generated under drought stress were involved in all algal events, including ABA biosynthesis, encystment, and enhanced carotenogenesis. These results indicate that ABA, induced by oxidative stress, could function as a stress hormone in algal morphogenesis in H. pluvialis under drought stress.     

Carotenogenesis in the green alga Haematococcus pluvialis: Cellular physiology and stress response

The unicellular green alga Haematococcus pluvialis Flotow has recently aroused considerable interest due to its capacity to amass large amounts of the ketocarotenoid astaxanthin (3,3'-dihydroxy- β , β -carotene-4,4'-dione), widely used commercially to color flesh of salmon. Astaxanthin accumulation in Haematococcus is induced by a variety of environmental stresses which limit cell growth in the presence of light. This is accompanied by a remarkable morphological and biochemical 'transformation' from green motile cells into inert red cysts. In recent years we have studied this transformation process from several aspects: defining conditions governing pigment accumulation, working out the biosynthetic pathway of astaxanthin accumulation and questioning the possible function of this secondary ketocarotenoid in protecting Haematococcus cells against oxidative damage. Our results suggest that astaxanthin synthesis proceeds via cantaxanthin and that this exceptional stress response is mediated by reactive oxygen species (ROS) through a mechanism which is not yet understood. The results do not support in vivo chemical quenching of ROS by the pigment, although in vitro it was shown to quench radicals very efficiently. The finding that most of the pigment produced is esterified and deposited in lipid globules outside the chloroplast further supports this assumption. We have suggested that astaxanthin is the by-product of a defense mechanism rather than the defending substance itself, although at this stage one cannot rule out other protective mechanisms. Further work is required for complete understanding of this transformation process. It is suggested that Haematococcus may serve as a simple model system to study response to oxidative stress and mechanisms evolved to cope with this harmful situation.     

Selective extraction of astaxanthin and chlorophyll from the green alga Haematococcus pluvialis

As astaxanthin could not be extracted with various organic solvents from intact thick-walled cyst cells of Haematococcus pluvialis, cells were held in 40% (v/v) acetone for 2 min at 80°C, followed by lyophilization or treatment of cells with specific lytic enzymes. By these treatments, 70% of the astaxanthin was extracted from the cells. Furthermore, the heat-acetone treatment was useful to remove chlorophyll specifically from the cells, contrary to saponification.     

Ketocarotenoid biosynthesis outside of plastids in the unicellular green alga Haematococcus pluvialis

The carotenoid biosynthetic pathway in algae and plants takes place within plastids. In these organelles, carotenoids occur either in a free form or bound to proteins. Under stress, the unicellular green alga Haematococcus pluvialis accumulates secondary carotenoids, mainly astaxanthin esters, in cytoplasmic lipid vesicles up to 4% of its dry mass. It is therefore one of the favored organisms for the biotechnological production of these antioxidative compounds. We have studied the cellular localization and regulation of the enzyme beta-carotene oxygenase in H. pluvialis that catalyzes the introduction of keto functions at position C-4 of the beta-ionone ring of beta-carotene and zeaxanthin. Using immunogold labeling of ultrathin sections and Western blot analysis of cell fractions, we discovered that under inductive conditions, beta-carotene oxygenase was localized both in the chloroplast and in the cytoplasmic lipid vesicles, which are (according to their lipid composition) derived from cytoplasmic membranes. However, beta-carotene oxygenase activity was confined to the lipid vesicle compartment. Because an early carotenogenic enzyme in the pathway, phytoene desaturase, was found only in the chloroplast (Grünewald, K., Eckert, M., Hirschberg, J., and Hagen, C. (2000) Plant Physiol. 122, 1261-1268), a transport of intermediates from the site of early biosynthetic steps in the chloroplast to the site of oxygenation and accumulation in cytoplasmic lipid vesicles is proposed.     

Transient expression of lacZ in bombarded unicellular green alga Haematococcus pluvialis

This paper reports for the first time the transient expression of areporter gene, LacZ, in the unicellular green alga Haematococcuspluvialis. By employing the micro-particle bombardment method,motilecells in the exponential phase showed transient expression oflacZ. This was detected in bombarded motile cells undertherupture-disc pressures of 3103 KPa and 4137 KPa.Transient expression of LacZ gene could not be observed in non-motile cells ofthis alga under the same transformation condition. No LacZ background was foundin either the motile cells or the non-motile cells. The study suggests apromising potential of the SV40 promoter and the lacZreporter gene in genetic engineering of unicellular green algae.     

Changes in pigments profile in the green alga Haeamtococcus pluvialis exposed to environmental stresses

Haematococcus green culture starved for either nitrogen or phosphate accumulated astaxanthin up to 4% cell dry wt (2.6 g l^–1). While under nitrogen starvation astaxanthin accumulation was faster (maximum achieved after 8 days in comparison to 14 days in the phosphate-starved culture) and accompanied by a drop in the chlorophyll content per cell down to 50% of its original value (30 pg cell^–1); in the phosphate-starved culture this parameter did not change. HPLC profiles of carotenoids monitored along the starvation process revealed that astaxanthin esters accounted for more than 99% of total carotenoids at the end of the exposure period at both starvations.     

Optimization of culture conditions for growth of the green alga Haematococcus pluvialis

The effects of light intensity, inoculum volume, sodium nitrate and carbon dioxide concentrations on the growth of Haematococcus pluvialis were investigated using response surface methodology (RSM). All the four variables exhibited significant effects on growth and can be related (r 0.926, P 0.01) by a second-order polynomial consisting of linear, quadratic and interaction terms. The total quadratic effect (P 0.01) dominates over the total linear effect (P 0.01) but the role of interaction terms (P 0.10) is marginal. The optimum values of these variables were: carbon dioxide 1.54%, sodium nitrate 1.06 g/l, inoculum volume 24.97% and light intensity 2.42 klux; the predicted maximum value for the yield of biomass was 0.51 g/l (dry weight).     

Astaxanthin production from the green alga Haematococcus pluvialis with different stress conditions

Summary Haematococcus pluvialis was induced to produce the astaxanthin pigment. A factorial design was carried out with three sodium acetate concentrations, 0.025, 0.05, 0.1 (g/l), and three NaCl concentrations (0.1, 0.2 and 0.4 %).The best conditions in algal culture for astaxanthin production were 0.2 % NaCl, 0.025 g/l sodium acetate and 0.05 g/l sodium acetate, each a 3.0, 1.83 and 1.78 % of astaxanthin, production in base to total dry weight, respectively. The higher astaxanthin production by bioreactor was 18.6 mg/l in the condition with 0.2 % NaCl.     

Singlet oxygen quenching ability of astaxanthin esters from the green alga Haematococcus pluvialis

The singlet oxygen quenching activities of carotenoids, -carotene, free astaxanthin, its monoester and its diester, were examined in vitro by a simple and rapid method for the measurement of Methylene Blue-sensitized photooxidation of linoleic acid in the hexane/ethanol solvent system. The concentrations of carotenoids, -carotene, free astaxanthin, its mono- and di-ester, required for 50% inhibition of lipid oxidation were 40, 8, 9, and 0 M in 100% ethanol and, 14, 16, 10, and 7 M in 50% (v/v) hexane in ethanol, respectively. Astaxanthin esters function as powerful antioxidant agents under both hydrophobic and hydrophilic conditions.     

Two components of photoreceptor potential in phototaxis of the flagellated green alga Haematococcus pluvialis

The kinetics of the photoreceptor potential of phototaxis in biflagellated green alga Haematococcus pluvialis in response to a 10-ns laser pulse of three wavelengths (465, 550, and 590 nm) were measured in single cells with 30 μs time resolution. The rise and the decay of photoinduced potential are both at least biphasic. The first component of the rise is very stable and has no measurable (<30 μs) time delay. The second component is triggered after a 120-400-μs lag period, depending on flash intensity. Its appearance is sensitive to the physiological state of the cell and the amplitude can be increased by phototactically ineffective red background illumination. The electrical generators for both components are localized in the same region of the cell membrane (on the stigma-bearing side) and these components have the same depolarizing sign. The results indicate that the photoreceptor potential in phototaxis comprises two components, which could be interpreted as light-induced charge movement within the photoreceptor molecules and changes in ion permeability of the cell membrane.     

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     

Stress-related differential expression of multiple beta-carotene ketolase genes in the unicellular green alga Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis is used as a biological production system for astaxanthin. It accumulates large amounts of this commercially interesting ketocarotenoid under a variety of environmental stresses. Here we report the identification and expression of three different beta-carotene ketolase genes (bkt) that are involved in the biosynthesis of astaxanthin in a single strain of the alga. Bkt1 and bkt2 proved to be the crtO and bkt previously isolated from two different strains of H. pluvialis. Bkt3 is a novel third gene, which shared 95% identical nucleotide sequence with bkt2. Nitrogen deficiency alone could not induce the alga cells to produce astaxanthin in 3 days even though it enhances the expression of the bkt genes to three times of that in normal growing cells within 16 h. High light irradiation (125 micromol m(-2)s(-1)) or 45 mM sodium acetate greatly increased the expression of bkt genes to 18 or 52 times of that in normal growing cells, resulting in an accumulation of substantial astaxanthin (about 6 mg g(-1) dry biomass) in 3 days. It is suggested that the existence of the multiple bkt genes and their strong up-regulation by different stress conditions is one of the reasons that H. pluvialis accumulates large amounts of astaxanthin in an instant response to stress environments.     

Selective loss of photosystem I and formation of tubular thylakoids in heterotrophically grown red alga Cyanidioschyzon merolae

Photosynthesis Research - We previously found that glycerol is required for heterotrophic growth in the unicellular red alga Cyanidioschyzon merolae. Here, we analyzed heterotrophically grown cells...