Green Alga Scenedesmus Obliquus Research Articles

A Novel Type of Iron Hydrogenase in the Green AlgaScenedesmus obliquus Is Linked to the Photosynthetic Electron Transport Chain

Hydrogen evolution is observed in the green alga Scenedesmus obliquus after a phase of anaerobic adaptation. In this study we report the biochemical and genetical characterization of a new type of iron hydrogenase (HydA) in this photosynthetic organism. The monomeric enzyme has a molecular mass of 44.5 kDa. The complete hydA cDNA of 2609 base pairs comprises an open reading frame encoding a polypeptide of 448 amino acids. The protein contains a short transit peptide that routes the nucleus encoded hydrogenase to the chloroplast. Antibodies raised against the iron hydrogenase from Chlamydomonas reinhardtii react with both the isolated and in Escherichia coli overexpressed protein of S. obliquus as shown by Western blotting. By analyzing 5 kilobases of the genomic DNA, the transcription initiation site and five introns within hydA were revealed. Northern experiments suggest that hydA transcription is induced during anaerobic incubation. Alignments of S. obliquus HydA with known iron hydrogenases and sequencing of the N terminus of the purified protein confirm that HydA belongs to the class of iron hydrogenases. The C terminus of the enzyme including the catalytic site (H cluster) reveals a high degree of identity to iron hydrogenases. However, the lack of additional Fe-S clusters in the N-terminal domain indicates a novel pathway of electron transfer. Inhibitor experiments show that the ferredoxin PetF functions as natural electron donor linking the enzyme to the photosynthetic electron transport chain. PetF probably binds to the hydrogenase through electrostatic interactions.

Electron pathways involved in H2-metabolism in the green alga Scenedesmus obliquus

The green alga Scenedesmus obliquus is capable of both uptake and production of H2 after anaerobic adaptation (photoreduction of CO2 or photohydrogen production). The essential enzyme for H2-metabolism is a NiFe-hydrogenase with a [2Fe–2S]-ferredoxin as its natural redox partner. Western blot analysis showed that the hydrogenase is constitutively expressed. The Km values were 79.5 μM and 12.5 μM, determined with ferredoxin and H2, respectively, as electron donor for the hydrogenase. In vitro, NADP+ was reduced by H2 in the presence of the hydrogenase, the ferredoxin and a ferredoxin–NADP reductase. From these results and considerations on the stoichiometry we propose that this light-independent electron transfer is part of the photoreduction of CO2 in vivo. For ATP synthesis, necessary for the photoreduction of CO2, light-dependent cyclic electron transfer around Photosystem (PS) I accompanies this ‘dark reaction’. PS II fluorescence data suggest that (a) in S. obliquus H2-reduction might function as the anaerobic counterpart of the O2-dependent Mehler reaction, and (b) the presence of either a ferredoxin quinone-reductase or NAD(P)-dehydrogenase (complex I) in S. obliquus chloroplasts.

DNA sequence analysis of the complete mitochondrial genome of the green alga Scenedesmus obliquus: evidence for UAG being a leucine and UCA being a non-sense codon

The complete DNA sequence of the mitochondrial genome of the chlorophyceen alga Scenedesmus obliquus was determined. The circular genome of 42 781 bp contains a basic set of 13 mitochondrial genes, which are conserved among plant or algal chondriomes. In addition, two scrambled rRNA and 27 tRNA genes are present, together with four intronic sequences (group I and II) and five open reading frames (ORFs), which show no significant homology to other ORFs from organellar genomes. The comparison with deduced amino acid sequences from 13 conserved mitochondrial genes gives rise to the conclusion that two deviations from the standard genetic code must be present in S. obliquus mitochondria: (i) UAG codes for leucine as was already found in some other algal mitochondria; (ii) UCA is a stop codon, which seems unique for mitochondrial genomes. This was supported by our finding that a tRNA-Leu gene possesses a UCA anticodon and by a missing tRNA-serine, able to decode the UCA codon. Consistent with these data is the absence of any UCA codon from conserved mitochondrial ORFs. This codon occurs only close to the end of all ORFs, while UAA or UGA codons are found at some distance from any conserved ORF. Codon changes by RNA editing can be excluded, since RT–PCR analysis does not reveal any evidence for post-transcriptional RNA modifications of the primary transcript.

Evidence for the Non-involvement of Nitric Oxide Synthase in Nitric Oxide Production by the Green Alga Scenedesmus obliquus

Studies conducted with N W -nitro-L-arginine (L-NNA) and N G -nitro-L-arginine-methyl ester (L-NAME), the potent inhibitors of nitric oxide synthase (NOS), did not exhibit any inhibitory effect on NO release by Scenedesmus cells grown either under illumination or in the dark supplemented with glucose. Further, these inhibitors did not interfere with the appearance of ‘light-off’ peaks, and the NO peaks also commenced following treatment with Cu, DCMU and glutarate in the presence of L-NNA and L-NAME. Complete suppression of NO production and the elimination of the most common ‘light-off’ peak were observed in algal culture grown in the medium where L-arginine was substituted for NO − 3 . This study therefore demonstrates that L-arginine is neither the substrate for NO production by Scenedesmus , as generally found in the case of animals and some plants, nor is the NOS involved in the nitric oxide production process.

ADAPTATION OF SCENEDESMUS OBLIQUUS (CHLOROPHYCEAE) TO COPPER‐DEFICIENCY: TRANSCRIPTIONAL REGULATION OF PCY1 BUT NOT CPX1

In contrast to the situation in Chlamydomonas reinhardtii, plastocyanin expression in the green alga Scenedesmus obliquus (Turp.) Kütz. (Chlorophyceae) is regulated by copper ion availability at the level of both protein and mRNA accumulation. To study the mechanism of copper‐dependent mRNA accumulation, plastocyanin‐encoding genomic and cDNA clones have been isolated and sequenced. The single nuclear S. obliquus Pcy1 gene contains two introns and a bipartite transit peptide sequence typical of plastid lumen‐localized proteins. Despite the similarity of the S. obliquus and Chlamydomonas reinhardtii Pcy1 genes, the S. obliquus Pcy1 gene was not expressed from its own promoter, nor could the S. obliquus Pcy1 promoter drive expression of a C. reinhardtii gene when it was introduced into the genome of C. reinhardtii cells. Using the cDNA clone as a probe, copper‐responsive regulation of Pcy1 expression was further characterized in S. obliquus. Experiments examining the time course of Pcy1 degradation on depletion of copper argue against a specific mechanism for Pcy1 mRNA decay in S. obliquus, suggesting that the copper‐responsive change in abundance occurs through transcriptional regulation. The Cpx1 gene (encoding coproporphyrinogen III oxidase) also responds differently to changes in cellular copper status in S. obliquus compared to C. reinhardtii. Whereas Cpx1 is highly induced in copper‐deficient cultures of C. reinhardtii, in S. obliquus the Cpx1 gene is not copper responsive.

Influence of selenium on toxicity of some heavy metals in the green alga Scenedesmus obliquus.

The green alga Scenedesmus obliquus was incubated with heavy metals (Cd2+, Zn2+, Mn2+, Ni2+) with and without selenium. S. obliquus exhibited higher rates of growth and some metabolic activities in cultures containing 0.1 mmol/L Se than those only containing the heavy metals. The positive effect of Se was found with all metals but was negligible with Mn2+.

Redox control of hydrogenase activity in the green alga Scenedesmus obliquus by thioredoxin and other thiols

The activity of the NiFe-hydrogenase from the green alga Scenedesmus obliquus is inhibited by both algal thioredoxins f and I+II, and by Escherichia coli thioredoxin. The strongest inhibition was observed with homologous chloroplastic thioredoxin f (I 50=21 nM) and E. coli thioredoxin (I 50=83 nM). For the homologous cytoplasmic thioredoxins I+II an I 50 of 667 nM was determined. Glutathione shows a similar but much less pronounced inhibitory effect whereas dithiothreitol had no effect. In addition to glucose-6-phosphate dehydrogenase, NiFe-hydrogenase is only the second enzyme known to be inhibited by reduced thioredoxin.

Amino acid sequence, crystallization and structure determination of reduced and oxidized cytochrome c6 from the green alga Scenedesmus obliquus

Cytochrome c 6 from the unicellular green alga Scenedesmus obliquus was sequenced, crystallized in its reduced and oxidized state and the three-dimensional structure of the protein in both redox states was determined by X-ray crystallography. Reduced cytochrome c 6 crystallized as a monomer in the space group P2 12 12, whereas the oxidized protein crystallized as a dimer in the space group P3 121. The structures were solved by molecular replacement and refined to 1.9 and 2.0 Å, respectively. Comparison of the structures of both redox states revealed only slight differences on the protein surface, whereas a distortion along the axis between the heme iron and its coordinating Met61 residue was observed. No redox-dependent movement of internal water molecules could be detected. The high degree of similarity of the surfaces and charge distributions of both redox states, as well as the dimerization of cytochrome c 6 as observed in the oxidized crystal, is discussed with respect to its biological relevance and its implications for the reaction mechanisms between cytochrome c 6 and its redox partners. The dimer of oxidized cytochrome c 6 may represent a molecular structure occurring in a binary complex with cytochrome b 6 f. This assembly might be required for the correct orientation of cytochrome c 6 with respect to its redox partner cytochrome b 6 f, facilitating the electron transfer within the complex. If the dimerization is not redox-dependent in vivo, the almost identical surfaces of both redox states do not support a long range differentiation between reduced and oxidized cyt c 6, i.e. a random collision model for the formation of an electron transfer complex must be assumed.

The regulatory role of polyamines in structure and functioning of the photosynthetic apparatus during photoadaptation

In this contribution we describe the changes in structure and functioning of the photosynthetic apparatus of the unicellular green alga Scenedesmus obliquus induced by inhibition or induction of polyamine biosynthesis. The synthesis or inhibition is controlled by white light of low and high intensities, as well as by blue and red irradiation. We observe that a decrease of the intracellular putrescine level and, vice versa, an increase of spermine, indicated by a raised ratio of spermine to putrescine, simulate a low-light photoadapted photosynthetic apparatus. Action spectra of the spermine/putrescine ratio compared with action spectra on characteristics of photoadaptation, e.g., chlorophyll biosynthesis, or the rate of primary photochemistry and electron transport, show that the photoreceptors for both the adaptation of the photosynthetic apparatus to low-light conditions and for the regulation of the spermine/putrescine ratio are, at least, very similar. We show that the photoreceptor is primarily a blue-light receptor with a superimposed red-light receptor that absorbs around 680 nm.

In vitro and in vivo coupling of Thiocapsa hydrogenase with cyanobacterial and algal electron mediators

H 2 activation by the oxygen-tolerant hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina, using electron mediators of cyanobacterial and algal origin, has been demonstrated. Ferredoxins, either from the cyanobacterium Synechococcus PCC7942 or the green alga Scenedesmus obliquus, are capable of providing electrons for hydrogenase-mediated H 2 evolution. The high-potential cytochrome c 6 from Synechococcus PCC7942 proved to be capable of accepting electrons derived from hydrogenase-mediated H 2 oxidation. In subsequent experiments, Thiocapsa hydrogenase was introduced into the cells of Synechococcus PCC7942 by electroporation, for enhanced H 2 production.

Group II intron splicing in chloroplasts: identificationof mutations determining intron stability and fate of exon RNA.

In order to investigate in vivo splicing of group II introns in chloroplasts, we previously have integrated the mitochondrial intron rI1 from the green alga Scenedesmus obliquus into the Chlamydomonas chloroplast tscA gene. This construct allows a functional analysis of conserved intron sequences in vivo, since intron rI1 is correctly spliced in chloroplasts. Using site-directed mutagenesis, deletions of the conserved intron domains V and VI were performed. In another set of experiments, each possible substitution of the strictly conserved first intron nucleotide G1 was generated, as well as each possible single and double mutation of the tertiary base pairing gamma-gamma ' involved in the formation of the intron's tertiary RNA structure. In most cases, the intron mutations showed the same effect on in vivo intron splicing efficiency as they did on the in vitro self-splicing reaction, since catalytic activity is provided by the intron RNA itself. In vivo, all mutations have additional effects on the chimeric tscA -rI1 RNA, most probably due to the role played by trans -acting factors in intron processing. Substitutions of the gamma-gamma ' base pair lead to an accumulation of excised intron RNA, since intron stability is increased. In sharp contrast to autocatalytic splicing, all point mutations result in a complete loss of exon RNA, although the spliced intron accumulates to high levels. Intron degradation and exon ligation only occur in double mutants with restored base pairing between the gamma and gamma' sites. Therefore, we conclude that intron degradation, as well as the ligation of exon-exon molecules, depends on the tertiary intron structure. Furthermore, our data suggest that intron excision proceeds in vivo independent of ligation of exon-exon molecules.

Comparison of light induced and cell cycle dependent changes in the photosynthetic apparatus: A fluorescence induction study on the green alga Scenedesmus obliquus

Changes in the photosynthetic apparatus occurring during the synchronous cell cycle of the green alga Scenedesmus obliquus are compared to the adaptational response induced by light intensity variations. To investigate and compare these two phenomena, we analyze the polyphasic rise of the chlorophyll fluorescence yield exhibited by plants and cyanobacteria when exposed to high intensity actinic light. Four fluorescence parameters are calculated which are closely related to Photosystem II (PS II) structure and function: ABS/RC, the antenna size of PS II; ϕPO, the quantum yield for reduction of the primary PS II quinone acceptor; qPQ, related to the size of the plastoquinone pool; qEmax, the capacity for pH dependent non-photochemical quenching. The capacity for non-photochemical quenching changes in response to light intensity variations, but it is not affected by the developmental changes occurring during the cell cycle. In contras t, for ABS/RC, ϕPO and qPQ, we observe light induced as well as cell cycle dependent variations. We discuss the relations of the four fluorescence parameters to the molecular organization of the photosynthetic apparatus and its cell cycle and light dependent changes.

The entire metabolite spectrum of the green alga Scenedesmus obliquus in isotope-labelled form

Abstract Modern biological structural and biosynthetic investigations often require the use of suitably isotope-labelled pigments or putative metabolic precursors. We present here, a culture and extraction protocol for Scenedesmus obliquus, which allows the simultaneous isolation of the-entire low M+ primary and secondary metabolite spectrum of this green alga. Fractions of amino acids, carbohydrates, lipids, carotenoids and chlorophylls are obtained, labelled in any combination of 2H, 13C and 15N.

Differential Changes in the Photosynthetic Pigments and Polyamine Content during Photoadaptation and Photoinhibition in the Unicellular Green Alga Scenedesmus obliquus

In the unicellular green alga Scenedesmus obliquus the level of photoinhibition and the recovery of the cells after reversal to the initial light conditions in relation to the pre-photoadaptation of the culture to low, medium and high light intensity was determined. The changes in the photosynthetic pigment content and in the intracellular polyamine concentration allowed to distinguish between photoadaptation and photoinhibition. In particular, the level of chlorophylls, xanthophylls and carotenoids decreased inversely proportional to the light intensity applied during photoadaptation, whereas their concentrations remained constant during photoinhibition. The violaxanthin/zeaxanthin and the loroxanthin/lutein cycle work only under photoinhibitory conditions, but not under photoadaptive premises. Changes in the level of these carotenoids in relation to the changes in the photosynthetic apparatus during photoadaptation are discussed. In addition, it was found that the intracellular polyamine level increased only under stress conditions, i. e. during photoinhibition, and decreased during recovery of the cells after reversal to the initial light conditions. The increase of the putrescine level during photoinhibition is inversely proportional to the light intensity used for pre-adaptation. This rise of the polyamine level in the cells photoadapted to high light conditions is an additional indication for the finding that photoadaptation and photoinhibition are different phenomena which are clearly distinguishable from each other. Finally, the changes of the chlorophyll, violaxanthin, zeaxanthin, loroxanthin, lutein and polyamine levels under photoadaptation in high light intensity (50 W m -2) in relation to the range of photoadaptation in Scenedesmus obliquus are discussed.

Food quality of detritus derived from the filamentous cyanobacterium Oscillatoria limneticafor Daphnia galeata

Detritus derived from the filamentous cyanobacterium , Oscillatoria limnetica was fed to Daphnia galeata Detritus supported growth and reproduction comparable to that on the green alga Scenedesmus obliquus . The live filaments of O.limnetica were, however, of lower food quality. Biochemical parameters of these food types, thought to be important in Daphnia nutrition, were also determined. It is concluded that detritus can be a good quality food for Daphnia , making eutrophic lakes better food environments than generally assumed.

On the long-wavelength spectral forms of chlorophyll a in Photosystem I: Spectroscopic and immunological investigations on a greening mutant of the green alga Scenedesmus obliquus

The origin of the long-wavelength chlorophyll (Chl) absorption (λpeak > 680 nm) and fluorescence emission (λpeak > 685 nm) has been investigated on Scenedesmus mutants (C-2A′-series, lacking the ability to synthesize chlorophyll in the dark) grown at 0.3 (LL), 10 (ML) and 240 µE s−1 m−2(HL). LL cells are arrested in an early greening state; consequently, ‘Chl availability’ determines the phenotype. LL thylakoids are totally lacking long-wavelength Chl; nonetheless, PS I and PS II are fully functional. Gel electrophoresis and Western blots indicate that four out of seven resolved LHC polypeptides seem to require a high Chl availability for assembly of functional chlorophyll-protein complexes. The PS I core-complex of ML and HL thylakoids contains long-wavelength chlorophylls, but in the PS I core-complex of LL thylakoids these pigments are lacking. We conclude that long-wavelength pigments are only present in the PS I core in the case of high Chl availability. The following hypothesis is discussed: Chl availability determines not only the LHC polypeptide pattern, but also the number of bound Chl molecules per individual pigment-protein complex. Chl-binding at non-obligatory, peripheral sites of the pigment-protein complex results in long-wavelength Chl. In the case of low Chl availability, these sites are not occupied and, therefore, the long-wavelength Chl is absent.

Effects of food type on the life history of Daphnia clones from lakes differing in trophic state. II. Daphnia cucullata feeding on mixed diets*

1. The effects of feeding on suboptimal foods were investigated in Daphnia cucullata a zooplankton common in many types of lakes. Eleven clones of D. cucullata were collected from four lakes of varying trophic levels and fed a high (1 mg C l–1) concentration of one of two diets: (i) a 1 : 9 mixture of the cryptophyte Cryptomonas pyrenoidifera with the green alga Scenedesmus obliquus; and (ii) a 1 : 9 mixture of the cryptophyte C. pyrenoidifera with the filamentous cyanobacterium Oscillatoria limnetica.2. As expected, the diet dominated by Oscillatoria was found to be significantly poorer compared with the diet dominated by Scenedesmus. All D. cucullata clones were, however, able to achieve positive population growth rates on both diets, supporting the view that daphnids can grow and reproduce on a diet dominated by filamentous cyanobacteria.3. Clones originating from different types of lakes differed in their responses to the two diets. On the Scenedesmus‐dominated diet, clones originating from moderately eutrophic lakes had lower population growth rates than clones from hypertrophic lakes. In contrast, on the Oscillatoria‐dominated diet, the clones from moderately eutrophic lakes had higher population growth rates than clones from hypertrophic lakes. Contrary to expectation, clones originating from lakes dominated by filamentous cyanobacteria had more difficulty utilizing filamentous cyanobacteria as food than clones from less eutrophic lakes which contain less filamentous cyanobacteria.4. As the reactions of clones originating from same type of lake resembled each other, it is hypothesized that the life histories of D. cucullata populations are locally adapted to environmental factors other than abundance of filamentous cyanobacteria, which are correlated with trophic levels.

Effects of food type on the life history of Daphnia clones from lakes differing in trophic state. I. Daphnia galeata feeding on Scenedesmus and Oscillatoria*

1. Effects of the green alga Scenedesmus obliquus and the filamentous cyanobacterium Oscillatoria limnetica as food sources on the growth and reproduction of nine Daphnia galeata clones were studied. A high concentration of these two algae species was fed to Daphnia clones collected from four lakes with varying trophic conditions in a life history experiment.2. Although Oscillatoria is regarded as a low quality food for Daphnia because of its filamentous morphology and suspected nutritional inadequacy, the cyanobacterium sustained both body growth and reproduction in D. galeata. Growth and reproduction were, however, lower and the onset of reproduction was delayed on a diet of Oscillatoria compared to a diet of Scenedesmus. The intrinsic growth rate, r, for each D. galeata clone was positive on a diet of Oscillatoria, ranging from 0.06 to 0.16 mm day–1, whereas the intrinsic growth rates on a diet of Scenedesmus ranged from 0.27 to 0.34 mm day–1.3. The degree of growth depression on the Oscillatoria diet varied among clones. Clonal responses were correlated with body size; clones with larger body size decreased their growth and reproduction more on a diet of Oscillatoria. The responses of different clones from the same lake to the two diets resembled each other, irrespective of geography or trophic state of the lake.

Assembly of light harvesting complexes II (LHC-II) in the absence of lutein: A study on the α-carotenoid-free mutant C-2A′-34 of the green alga Scenedesmus obliquus

Stable assembly of the major LHC-II proteins (22, 26 kDa) is inhibited in the α-carotenoid-free mutant C-2A′-34 of the green alga Scenedesmus obliquus, whereas other LHC-II proteins (22, 23, 29 kDa) seem to be not affected. We conclude that lutein is essential for assembly of the major LHC-II proteins of S. obliquus, but not for assembly of the minor LHC-II proteins. It is proposed that in the minor LHC-II proteins lutein is replaced by violaxanthin, zeaxanthin and antheraxanthin.

Biotransformation of 1-naphthalenesulfonic acid by the green alga Scenedesmus obliquus.

Under sulfate limitation, axenic batch cultures of the green alga Scenedesmus obliquus metabolized 1-naphthalenesulfonic acid and partially used the sulfonate as a source of sulfur. The main metabolite, 1-hydroxy-2-naphthalenesulfonic acid, which was not metabolized further in the algal culture, was formed by hydroxylation of the substrate in position 1 and by migration of the sulfonic acid group to position 2 of the naphthalene ring (NIH shift). A smaller amount of 1-naphthalenesulfonic acid was desulfonated. The resulting 1-naphthol was mostly transformed into 1-naphthyl beta-d-glucopyranoside.