Aliza Zarka

ACCUMULATION OF OLEIC ACID IN HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE) UNDER NITROGEN STARVATION OR HIGH LIGHT IS CORRELATED WITH THAT OF ASTAXANTHIN ESTERS1

The chlorophyte Haematococcus pluvialis accumulates large quantities of astaxanthin under stress conditions. Under either nitrogen starvation or high light, the production of each picogram of astaxanthin was accompanied by that of 5 or 3–4 pg of fatty acids, respectively. In both cases, the newly formed fatty acids, consisting mostly of oleic (up to 34% of fatty acids in comparison with 13% in the control), palmitic, and linoleic acids, were deposited mostly in triacylglycerols. Furthermore, the enhanced accumulation of oleic acid was linearily correlated with that of astaxanthin. Astaxanthin, which is mostly monoesterified, is deposited in globules made of triacylglycerols. We suggest that the production of oleic acid‐rich triacylglycerols on the one hand and the esterification of astaxanthin on the other hand enable the oil globules to maintain the high content of astaxanthin esters.

ASTAXANTHIN ACCUMULATION IN HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE) AS AN ACTIVE PHOTOPROTECTIVE PROCESS UNDER HIGH IRRADIANCE1

Green cells of Haematococcus pluvialis Flotow accumulate the ketocarotenoid astaxanthin under stress conditions, such as high irradiance, nutrient deficiency, high salinity, and high temperature. Though some photoprotective mechanisms have been suggested, the function of astaxanthin in red cysts is still questioned. We studied the role of astaxanthin in photoprotection by inducing its formation in logarithmically growing cultures by high irradiance, thus avoiding unrelated processes that can occur in H. pluvialis when carotenogenesis is induced by other stresses. On exposure to high irradiance, the green Haematococcus culture turned red as lipid globules loaded with astaxanthin esters were formed and concentrated at the periphery of the cell. During this phase of induction, the photosynthesis rates remained high, but the amount of the D1 protein of PSII was significantly reduced. The decline in D1 protein content stopped after 1 day; the level then increased, returning to normal after 5 days. The response of the D1 protein was indicative of a transitional phase in the acclimation of Haematococcus to high light. The formation and deposition of astaxanthin seemed to prevent further reduction in D1 protein level, thus enabling the cell to maintain PSII function and structural integrity. This result seems to be a clear indication of the light screening by astaxanthin, which absorbs light in the blue region, thus protecting the photosynthetic apparatus. When the cells recovered from the high light stress, the astaxanthin globules concentrated around the nucleus, indicating that the pigment also serves as a physicochemical barrier, protecting the replicating DNA from oxidation as the cells divide.

Isolation and characterization of a novel chytrid species (phylum Blastocladiomycota), parasitic on the green alga Haematococcus.

A parasite was found in cultures of the green microalga Haematococcus pluvialis that grew epibiotically on algal cells and caused epidemics resulting in damage to the host cultures. The parasite was isolated into axenic culture on solid and liquid media. It was demonstrated to be the sole causative agent of the epidemics. According to its life cycle and phylogenetic analysis based on 18S ribosomal DNA sequences, the pathogen appears to represent a novel chytrid fungus closely related to the vascular plant pathogen Physoderma (Blastocladiomycota), although it differs from all other known chytrids by its infective stage, a wall-less propagule endowed with amoeboid motion and lacking the groups typical flagellum.

INHIBITION OF ASTAXANTHIN SYNTHESIS UNDER HIGH IRRADIANCE DOES NOT ABOLISH TRIACYLGLYCEROL ACCUMULATION IN THE GREEN ALGA HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE) 1

Under stress conditions, Haematococcus pluvialis Flotow accumulates fatty acid–esterified astaxanthin, in extraplastidial lipid globules. The enhanced accumulation of fatty acids, mainly in triacylglycerols (TAG), among which oleic acid predominates, is linearly correlated with that of astaxanthin. We used inhibitors of either carotenoid or lipid biosynthesis to assess the interrelationship between carotenogenesis and TAG accumulation under high light irradiance as the stress factor. The two carotenogenesis inhibitors used—norflurazon, an inhibitor of phytoene desaturase, and diphenylamine (DPA), an inhibitor of β‐carotene C‐4 oxygenase—suppressed the accumulation of astaxanthin in a concentration‐dependent manner. Concurrently, the accumulation of neutral lipids was significantly less affected. The lipid biosynthesis inhibitor sethoxydim, which inhibits acetyl‐CoA carboxylase, significantly decreased de novo fatty acid synthesis and, in concert, drastically inhibited astaxanthin formation. In the presence of various concentrations of the three inhibitors, the inhibition of astaxanthin was not accompanied by a proportional decrease in oleic acid, which was used as a marker for TAG fatty acids. When astaxanthin synthesis was completely inhibited, the volumetric content of oleic acid was about 60% of the control value when the two carotenogenesis inhibitors (0.05 μM norflurazon or 20 μM DPA) were used and 27% of the control when the lipid‐synthesis inhibitor (50 μM) was used. We suggest therefore that TAG accumulation under high irradiance is not tightly coupled with astaxanthin accumulation, although the correlation between these two processes was demonstrated earlier. Furthermore, we propose that the accumulation of a certain amount of TAG is a prerequisite for the initiation of fatty acid–esterified astaxanthin accumulation in lipid globules.

Cloning and molecular characterization of a novel acyl‐CoA:diacylglycerol acyltransferase 1‐like gene (PtDGAT1) from the diatom Phaeodactylum tricornutum

We have identified and isolated a cDNA encoding a novel acyl‐CoA:diacylglycerol acyltransferase (DGAT)1‐like protein, from the diatom microalga Phaeodactylum tricornutum (PtDGAT1). The full‐length cDNA sequences of PtDGAT1 transcripts revealed that two types of mRNA, PtDGAT1short and PtDGAT1long, were transcribed from the single PtDGAT1 gene. PtDGAT1short encodes a 565 amino acid sequence that is homologous to several functionally characterized higher plant DGAT1 proteins, and 55% identical to the putative DGAT1 of the diatom Thalassiosira pseudonana, but shows little homology with other available putative and cloned algal DGAT sequences. PtDGAT1long lacks several catalytic domains, owing to a 63‐bp nucleotide insertion in the mRNA containing a stop codon. Alternative splicing consisting of intron retention appears to regulate the amount of active DGAT1 produced, providing a possible molecular mechanism for increased triacylglycerol (TAG) biosynthesis in P. tricornutum under nitrogen starvation. DGAT mediates the last committed step in TAG biosynthesis, so we investigated the changes in expression levels of the two types of mRNA following nitrogen starvation inducing TAG accumulation. The abundance of both transcripts was markedly increased under nitrogen starvation, but much less so for PtDGAT1short. PtDGAT1 activity of PtDGAT1short was confirmed in a heterologous yeast transformation system by restoring DGAT activity in a Saccharomyces cerevisiae neutral lipid‐deficient quadruple mutant strain (H1246), resulting in lipid body formation. Lipid body formation was only restored upon the expression of PtDGAT1short, and not of PtDGAT1long. The recombinant yeast appeared to display a preference for incorporating saturated C16 and C18 fatty acids into TAG.

Does astaxanthin protect Haematococcus against light damage

Abstract The photoprotective function of the ketocarotenoid astaxanthin in Haematococcus was questioned. When exposed to high irradiance and/or nutritional stress, green Haematococcus cells turned red due to accumulation of an immense quantity of the red pigment astaxanthin. Our results demonstrate that: 1) The addition of diphenylamine, an inhibitor of astaxanthin biosynthesis, causes cell death under high light intensity; 2) Red cells are susceptible to high light stress to the same extent or even higher then green ones upon exposure to a very high light intensity (4000 μmol photon m-2 s-1); 3) Addition of 1O2 generators (methylene blue, rose bengal) under noninductive conditions (low light of 100 (μmol photon m-2 s-1) induced astaxanthin accumulation. This can be reversed by an exogenous 1O2 quencher (histidine); 4) Histidine can prevent the accumulation of astaxanthin induced by phosphate starvation. We suggest that: 1) Astaxanthin is the result of the photoprotection process rather than the protective agent; 2) 1O2 is involved indirectly in astaxanthin accumulation process.

A simplified protocol for preparing DNA from filamentous cyanobacteria

The preparation of good quality genomic DNA from microalgae and plants is often time-consuming because of the need to remove contaminants that may interfere with the downstream enzymatic manipulation of the DNA. Simpler protocols have been reported but these are applicable only to a few species and in many cases are not effective for removing trace contaminants. In this report, we describe a modification of existing protocols that significantly simplified the preparation of genomic DNA from cyanobacteria and plants. A key step in our protocol is the precipitation of DNA in a high concentration of salt (2–2.5 M NaCl) in the presence of isopropanol, immediately following phenol and chloroform extractions. The preparation and enzymatic digestion of the DNA can be performed in a single day. The DNA was easily digested in 2 h at normal restriction enzyme concentrations, and is highly suitable for PCR and Southern hybridization. We successfully used this simplified protocol to prepare genomic DNA from several filamentous cyanobacteria, such asAnabaena sp. PCC 7120,Anabaena siamensis, andSpirulina strains M2 and Kenya. This protocol may also be useful for preparing genomic DNA from other algae and from higher plants.

Presence of a Nonhydrolyzable Biopolymer in the Cell Wall of Vegetative Cells and Astaxanthin-Rich Cysts of Haematococcus pluvialis (Chlorophyceae)

Abstract: The green alga Haematococcus pluvialis accumulates massive amounts of the red pigment astaxanthin in response to stimuli inducing it to form cysts. During the encystment process the cell wall undergoes a clear hardening and thickening. In this work, a cell wall fraction withstanding successive acid and basic hydrolysis was isolated and proves to be algaenan by Fourier transform infrared spectroscopy. This compound is equally abundant in nonmotile vegetative cells and astaxanthin-rich cysts. This finding indicates that the synthesis of algaenan does not require the activation of the machinery for the massive production of secondary carotenoids. We conclude that algaenan cannot cause the changes occurring in the cell wall during the encystment process without the involvement of other cell wall components.

Metabolite Profiling and Integrative Modeling Reveal Metabolic Constraints for Carbon Partitioning under Nitrogen Starvation in the Green Algae Haematococcus pluvialis

Background: The microalga H. pluvialis shows a distinct pattern of carbon repartitioning upon nitrogen starvation. Results: Data-driven integrative modeling pinpoints metabolic adjustments underlying carbon repartition. Conclusion: In vitro and in silico experiments can dissect the systemic response to nitrogen starvation. Significance: Experimental data in conjunction with integrative modeling enables model-driven hypothesis testing. The green alga Hematococcus pluvialis accumulates large amounts of the antioxidant astaxanthin under inductive stress conditions, such as nitrogen starvation. The response to nitrogen starvation and high light leads to the accumulation of carbohydrates and fatty acids as well as increased activity of the tricarboxylic acid cycle. Although the behavior of individual pathways has been well investigated, little is known about the systemic effects of the stress response mechanism. Here we present time-resolved metabolite, enzyme activity, and physiological data that capture the metabolic response of H. pluvialis under nitrogen starvation and high light. The data were integrated into a putative genome-scale model of the green alga to in silico test hypotheses of underlying carbon partitioning. The model-based hypothesis testing reinforces the involvement of starch degradation to support fatty acid synthesis in the later stages of the stress response. In addition, our findings support a possible mechanism for the involvement of the increased activity of the tricarboxylic acid cycle in carbon repartitioning. Finally, the in vitro experiments and the in silico modeling presented here emphasize the predictive power of large scale integrative approaches to pinpoint metabolic adjustment to changing environments.

The host-range of Paraphysoderma sedebokerensis, a chytrid that infects Haematococcus pluvialis

A parasitic chytrid that attacks the green alga Haematococcus pluvialis was recently isolated in our laboratory and identified as a novel species from the phylum Blastocladiomycota, named herein Paraphysoderma sedebokerensis (nom. prov.). A method for early and precise detection of chytrid infections was developed using the fluorescent dye, Nile red, which stained the chytrids’ sporangia. Using this technique we determined the specificity of Paraphysoderma sedebokerensis for 13 algal species belonging to the Chlorophyta. Algal species tested including: Chlamydomonas nivalis, Chlorella emersonii, Chlorella vulgaris, Chlorococcum sp., Chlorogonium elongatum, Monoraphidium braunii, Muriella zofigiensis, Scenedesmus obliquus, Scenedesmus vacuolatus (two strains), and Scotiellopsis oocystiformis were either resistant to infection, or only experienced slight levels of infection during exponential growth. During exponential growth phase 100% Chlorella zofigiensis Donz cells were infected, but none developed any infection during resting stage. Only in cultures of H. pluvialis did infections develop rapidly (3–4 days) and intensively (100% cells infected) during both the logarithmic and stationary stages of growth. We suggest that the newly isolated chytrid, Paraphysoderma sedebokerensis (nom. prov.), is highly specific for H. pluvialis, but has a limited capacity to infect other green algae.