Identification of a gene for UDP-sulfoquinovose synthase of a green alga, Chlamydomonas reinhardtii, and its phylogeny.

Sulfoquinovosyl diacylglycerol is responsible for the structural and functional integrity of the photosystem II complex of a green alga, Chlamydomonas reinhardtii. We cloned a cDNA of C. reinhardtii containing an open reading frame for a protein 36-64% identical in the primary structure to known UDP-sulfoquinovose synthases, which are required for SQDG synthesis, in other organisms. Through the introduction of the cDNA, a cyanobacterial disruptant as to the UDP-sulfoquinovose synthase gene recovered the ability to synthesize sulfoquinovosyl diacylglycerol, thus confirming that the cDNA encodes the UDP-sulfoquinovose synthase. On the genome, the cDNA was divided into 14 exons, and the gene designated as SQD1 was present as one copy. The molecular phylogenetic tree for the UDP-sulfoquinovose synthase showed grouping of C. reinhardtii together with species that require sulfoquinovosyl diacylglycerol for the functioning of the PSII complex, but not with those that do not utilize the lipid for photosynthesis. The role of sulfoquinovosyl diacylglycerol in the functioning of the photosynthetic membranes might evolve in harmony with the system of the membrane lipid synthesis such as UDP-sulfoquinovose synthase gene.     

Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene （apcA and apcB） into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.     

Comparative proteomics of high light stress in the model alga Chlamydomonas reinhardtii

High light (HL) stress adversely affects growth, productivity and viability of photosynthetic organisms. The green alga Chlamydomonas reinhardtii is a model system to study photosynthesis and light stress. Comparative proteomics of wild-type and two very high light (VHL)-resistant mutants, VHL(R)-S4 and VHL(R)-S9, revealed complex alterations in response to excess light. A two-dimensional reference map of the soluble subproteome was constructed representing about 1500 proteins. A total of 83 proteins from various metabolic pathways were identified by peptide mass fingerprinting. Quantitative comparisons of 444 proteins showed 105 significantly changed proteins between wild type and mutants under different light conditions. Commonly, more proteins were decreased than increased, but different proteins were affected in each genotype. Proteins uniquely altered in either VHL(R) mutant may be involved in VHL resistance. Such candidate proteins similarly altered without light stress, thus possibly contributing to "pre-adaptation" of mutants to VHL, included decreased levels of a DEAD box RNA helicase (VHL(R)-S4) and NAB1 and RB38 proteins (VHL(R)-S9), and increased levels of an oxygen evolving enhancer 1 (OEE1) isoform and an unknown protein (VHL(R)-S4). Changes from increased levels in HL to decreased levels in excess light, included OEE1 (VHL(R)-S9) or the reverse change for NAB1, RB38, beta-carbonic anhydrase and an ABC transporter-like protein (VHL(R)-S4).     

Kinetic modeling of the photosynthetic growth of Chlamydomonas reinhardtii in a photobioreactor

The aim of this study was to establish and validate a model for the photosynthetic growth of Chlamydomonas reinhardtii in photobioreactors (PBRs). The proposed model is based on an energetic analysis of the excitation energy transfer in the photosynthesis apparatus (the Z-scheme for photosynthesis). This approach has already been validated in cyanobacteria (Arthorspira platensis) and is extended here to predict the volumetric biomass productivity for the microalga C. reinhardtii in autotrophic conditions, taking into consideration the two metabolic processes taking place in this eukaryotic microorganism, namely photosynthesis and respiration. The kinetic growth model obtained was then coupled to a radiative transfer model (the two-flux model) to determine the local kinetics, and thereby the volumetric biomass productivity, in a torus PBR. The model was found to predict PBR performances accurately for a broad set of operating conditions, including both light-limited and kinetic growth regimes, with a variance of less than 10% between experimental results and simulations.     

Dna insertional mutagenesis for the elucidation of a Photosystem II repair process in the green alga Chlamydomonas reinhardtii

The work outlines the isolation of transformant Chlamydomonas reinhardtii cells that appear to be unable to repair Photosystem II from photoinhibitory damage. A physiological and biochemical characterization of three mutants is presented. The results show differential stability for the D1 reaction center protein in the three mutants compared to the wild type and suggest lesions that affect different aspects of the Photosystem II repair mechanism. In the ag16.2 mutant, significantly greater amounts of D1 accumulate in the thylakoid membrane than in the wild type under steady-state growth conditions, and D1 loss is significantly retarded in the presence of the protein biosynthesis inhibitor chloramphenicol. Moreover, aberrant electrophoretic mobility of D1 in the ag16.2 suggests that this protein is modified to an as yet unknown configuration. These results indicate that the biosynthesis and/or degradation of D1 is altered in this strain. A different type of mutation occurred in the kn66.7 and kn27.4 mutants of C. reinhardtii. The stability of D1 declined much faster as a function of light intensity in these mutants than in the wild type. Thereby, the threshold of photoinhibition in these mutants was significantly lower than that in the wild type. It appears that kn66.7 and kn27.4 are similar conditional mutants, with the only difference between them being the amplitude of the chloroplast response to the mutation and the differential sensitivity they display to the level of irradiance.     

A large scale structural analysis of cDNAs in a unicellular green alga, Chlamydomonas reinhardtii. I. Generation of 3433 non-redundant expressed sequence tags.

To understand genetic information carried in a unicellular green alga, Chlamydomonas reinhardtii, normalized and size-selected cDNA libraries were constructed from cells at photoautotrophic growth, and a total of 11,571 5'-end sequence tags were established. These sequences were grouped into 3433 independent EST species. Similarity search against the public non-redundant protein database indicated that 817 groups showed significant similarity to registered sequences, of which 140 were of previously identified C. reinhardtii genes, but the remaining 2616 species were novel sequences. The coverage of full-length protein coding regions was estimated to be over 60%. These cDNA clones and EST sequence information will provide a powerful source for the future genome-wide functional analysis of uncharacterized genes.     

Ultrastructural,cytochemical and immunocytochemical investigation of the interphase nucleus in the unicellular green alga Chlamydomonas reinhardtii

Summary— The ultrastructural organization of the interphase nucleus of the green alga Chlamydomonas reinhardtii was investigated and found to be largely dependent on the fixation conditions. In specimens stained with bismuth, densely contrasted granules ranging from 25 to 45 nm in diameter were localized throughout the interchromatin space and often formed clusters. These granules were labeled by RNase A-gold complexes and may represent the counterparts of animal and higher plant cll interchromatin granules. Within the nucleolus the Ag-NOR and pyroantimonate stains and, to a lesser extent, the bismuth stain reacted with the nucleolar dense fibrillar component (DFC). When cells were subjected to a heat shock at 42°C, the nucleolar DFC was found to progressively separate from the nucleolus and, after 3 h, appeared as a continuous meandering thread about 0.1 μm in width. Within the nucleolus, labeling on conventional preparations occurred as small clusters with antibodies to H3 histones or to DNA whereas RNase A-gold complexes labeled most of it including fibrillar centers. Improved ultrastructural preservation in cryofixed, cryosubstituted specimens gently fixed in glutaraldehyde permitted to localize nucleolar DNA predominantly at the outer edge of fibrillar centers and to a lesser extent within the neighbouring DFC. Our results indicate that the structure and composition of Chlamydomonas interphase nuclei are comparable, despite particularities, to those of animal and higher plant nuclei.     

Composition of nuclear dense bodies and nucleolus-associated bodies in interphase nuclei of the unicellular green alga Chlamydomonas reinhardtii

Summary— The interphase nucleus of the green alga, Chlamydomonas reinhardtii, displayed two types of bodies some of them, the dense bodies, lying apparently free in the nucleoplasm while the others were attached to the nucleolus and were, therefore, referred to as nucleolus-associated bodies (NABs). The presence of DNA, RNA and histones in dense bodies was investigated by means of post-embedding immunocytochemistry and cytochemistry using a monoclonal antibody to single and double stranded DNA, a polyclonal antibody to rye H3 histones and RNase A-gold complexes. The dense bodies were shown to contain significant amounts of RNA but neither DNA nor histones were detected; their composition was thus similar to that of the dense bodies described in higher plant cells. We propose that dense bodies might be implicated in the assembly of the 25 to 45 nm granules observed throughout the nucleoplasm of Chalamydomonas interphase nuclei. The composition of NABs was found to be distinct from that of the dense bodies since they were labeled by the antibody to DNA, specially in cryofixed and cryosubstituted specimens. The presence of DNA in NABs together with their intimate association to the nucleolus suggest that they may correspond to specific segments of chromosomes.     

Temperature-dependent photoinhibition and recovery of photosynthesis in the green alga Chlamydomonas reinhardtii acclimated to 12 and 27°C

Photoinhibition of photosynthesis and subsequent recovery were studied in cultures of the unicellular green alga Chlamydomonas reinhardtii L. (wt strain 137 c mating type +) acclimated at high (27°C) and low (12°C) temperature, Photoinhibition was assayed by fluorescence kinetics (77K) and oxygen evolution measurements under growth temperature conditions Inhibition of 50% was obtained by exposing cultures acclimated at high temperature to a photosynthetic photon flux density (PPFD) of 1 600 μmol m⁻² S⁻¹ at. 27°C. and cultures acclimated at low temperature to a PPFD of 900 μmol m⁻² s⁻¹ at 12°C When the photoinhibitory conditions were shifted it was revealed that algae acclimated at low temperature had acquired an increased resistance to photoinhibition at both 12 and 27°C. Furthermore, acclimation at low temperature increased the capacity to recover from 50% photoinhibition at both 12 and 27°C Studies of photoinhibition in the presence of the protein synthesis inhibitor, chloramphenicol, revealed that in response to acclimation at low temperature during growth the algae became more dependent on protein synthesis to avoid photoinhibition. It is suggested that acclimation at low temperature rendered C. reinhardtii an increased resistance to photoinhibition by. increasing the rate of turnover of photodamaged proteins in photosystem II (PS II). However, we cannot exclude the possibility that the increased resistance to photoinhibition of C. reinhardtii acclimated at low temperature also involves modifications of the mechanism of photoinhibition.     

Molybdenum cofactor amounts in Chlamydomonas reinhardtii depend on the Nit5 gene function related to molybdate transport

Strain 21gr from Chlamydomonas reinhardtii is a cryptic mutant defective in the Nit5 gene related to the biosynthesis of molybdenum cofactor (MoCo). In spite of this mutation, this strain has active MoCo and can grow on nitrate media. In genetic crosses, the Nit5 mutation cosegregated with a phenotype of resistance to high concentrations of molybdate and tungstate. Molybdate/tungstate toxicity was much higher in nitrate than in ammonium media. Strain 21gr showed lower amounts of MoCo activity than the wild type both when grown in nitrate and after growth in ammonium and nitrate induction. However, nitrate reductase (NR) specific activity was similar in wild type and 21gr cells. Tungstate, either at nanomolar concentrations in nitrate media or at micromolar concentrations during growth in ammonium and nitrate induction, strongly decreased MoCo and NR amounts in wild‐type cells but had a slight effect in 21gr cells. Molybdate uptake activity of ammonium‐grown cells from both the wild‐type and 21gr strains was small and blocked by sulphate 0·3 mM . However, cells from nitrate medium showed a molybdate uptake activity insensitive to sulphate. This uptake activity was much higher and more sensitive to inhibition by tungstate in the wild type than in strain 21gr. These results suggest that strain 21gr has a high affinity and low capacity molybdate transport system able to discriminate efficiently tungstate, and lacks a high capacity molybdate/tungstate transport system, which operates in wild‐type cells upon nitrate induction. This high capacity molybdate transport system would account for both the stimulating effect of molybdate on MoCo amounts and the toxic effects of tungstate and molybdate when present at high concentrations.     

Programmed cell death is induced by hydrogen peroxide but not by excessive ionic stress of sodium chloride in the unicellular green alga Chlamydomonas reinhardtii

Eukaryotic microalgae serve as indicators of environmental change when exposed to severe seasonal fluctuations. Several environmental stress conditions are known to produce reactive oxygen species in cellular compartments, resulting in oxidative damage and apoptosis. The study of cell death in higher plants and animals has revealed the existence of an active ‘programmed cell death’ (PCD) process and similarities between such processes suggest an evolutionary origin. A study was undertaken to examine the morphological, biochemical and molecular responses of the unicellular green alga Chlamydomonas reinhardtii after exposure to oxidative (10 mM H₂O₂) and osmotic (200 mM NaCl and 360 mM sorbitol) stress. Concentrations of H₂O₂ (2–50 mM), NaCl and sorbitol (100–800 mM) were negatively correlated with growth. Biochemical analyses showed an increase in intracellular H₂O₂ production (2.2-fold with H₂O₂ and ~1.2–1.4-fold with NaCl and sorbitol) and activities of some antioxidant enzymes [super oxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX)]. Alteration of mitochondrial membrane potential (MMP) was observed upon treatment with H₂O₂ and NaCl, but not with sorbitol, indicating that the ionic stress component of NaCl altered the MMP. In addition, H₂O₂ led to the activation of a caspase-3-like protein, increase in the cleavage of a poly(ADP) ribose polymerase-1 (PARP-1)-like enzyme and formation of DNA nicks and laddering. With NaCl and sorbitol, no caspase activation, nor oligonucleosomal DNA laddering was observed, indicating non-apoptotic death. However, genomic DNA of NaCl (800 mM)-stressed cells, but not those of sorbitol-treated cells showed complete shearing. We conclude that the ionic rather than the osmotic component of NaCl leads to necrosis. These results unequivocally suggest that the vegetative cells of C. reinhardtii respond differentially to various stress agents, leading to different death types in the same organism. Moreover, unlike most other organisms, when exposed to NaCl this alga does not undergo PCD.     

Fate of substituted benzoates in the freshwater green alga, Chlamydomonas reinhardtii 11-32b

The removal of chlorinated, nitrated, and sulfonated benzoic acids in cultures of the unicellular green alga, Chlamydomonas reinhardtii 11-32b, was investigated, and the metabolic fate of a model compound, 4-chloro-3,5-dinitrobenzoic acid, was determined. The freshwater alga was able to remove a wide variety of benzoic compounds from the incubation medium. Chlamydomonas discriminated very specifically between the benzoic acids, indicated by the varying degrees of which the test compounds disappeared from the culture medium. Moreover, the alga was capable of transforming 4-chloro-3,5-dinitrobenzoic acid to several metabolites. A release of chloride ions was observed, and 3,5- dinitro-4-hydroxybenzoic acid was identified as a major transient product in the algal metabolism of 4-chloro-3,5-dinitrobenzoic acid.     

Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii

The basic question addressed in this study is how energy metabolism is adjusted to cope with iron deficiency in Chlamydomonas reinhardtii. To investigate the impact of iron deficiency on bioenergetic pathways, comparative proteomics was combined with spectroscopic as well as voltametric oxygen measurements to assess protein dynamics linked to functional properties of respiratory and photosynthetic machineries. Although photosynthetic electron transfer is largely compromised under iron deficiency, our quantitative and spectroscopic data revealed that the functional antenna size of photosystem II (PSII) significantly increased. Concomitantly, stress-related chloroplast polypeptides, like 2-cys peroxiredoxin and a stress-inducible light-harvesting protein, LhcSR3, as well as a novel light-harvesting protein and several proteins of unknown function were induced under iron-deprivation. Respiratory oxygen consumption did not decrease and accordingly, polypeptides of respiratory complexes, harboring numerous iron-sulfur clusters, were only slightly diminished or even increased under low iron. Consequently, iron-deprivation induces a transition from photoheterotrophic to primarily heterotrophic metabolism, indicating that a hierarchy for iron allocations within organelles of a single cell exists that is closely linked with the metabolic state of the cell.     

Short Communication: Analysis of effective light in different photobioreactors: its influence on growth,photosynthetic activity and glycerol production by the freshwater green alga Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii synthesizes glycerol as an osmoregulatory metabolite when exposed to high saline concentrations (200 mM NaCl). Response to osmotic stress can be used for biotechnological production of this compound. When synthesis of a substance is linked to photosynthetic capacity and consequently to effective light, the production on a large scale makes an efficient utilization of light necessary. In the present work a model for evaluation of effective light has been tested.     

Ligation-mediated suppression-PCR as a powerful tool to analyse nuclear gene sequences in the green alga Chlamydomonas reinhardtii

To improve the analysis of unknown flanking DNA sequences adjacent to known sequences in nuclear genomes of photoautotrophic eukaryotic organisms, we established the technique of ligation-mediated suppression-PCR (LMS-PCR) in the green alga Chlamydomonas reinhardtii for (1) walking from a specific nuclear insertion fragment of random knockout mutants into the unknown flanking DNA sequence to identify and analyse disrupted genomic DNA regions and for (2) walking from highly conserved DNA regions derived from known gene iso-forms into flanking DNA sequences to identify new members of protein families. The feasibility of LMS-PCR for these applications was successfully demonstrated in two different approaches. The first resulted in the identification of a genomic DNA fragment flanking a nuclear insertion vector in a random knockout mutant whose phenotype was characterised by its inability to perform functional LHC state transitions. The second approach targeted the cab gene family. An oligonucleotide of a cabII gene, derived from a highly conserved region, was used to identify potential cab gene regions in the nuclear genome of Chlamydomonas. LMS-PCR combined with 3′ rapid amplification of cDNA ends (3′ RACE) and a PCR-based screening of a cDNA library resulted in the identification of the new cabII gene lhcb4. Both results clearly indicate that LMS-PCR is a powerful tool for the identification of flanking DNA sequences in the nuclear genome of Chlamydomonas reinhardtii. This revised version was published online in June 2006 with corrections to the Cover Date.     

The role of extracellular carbonic anhydrase for accumulation of inorganic carbon in the green alga Chlamydomonas reinhardtii. A comparison between wild-type and cell-wall-less mutant cells

The role of extracellular carbonic anhydrase (CA_ex) for dissolved inorganic carbon (DIC) accumulation in the green alga Chlamydomonas reinhardtii was investigated. It was found that when algal cells were bubbled with ambient air, cell-wall-less mutant cells exhibited the same high photosynthetic affinity for CO₂ as wild-type cells despite a 10 times lower activity of CA^ex. It was also found that the affinity for CO₂ was further increased when the total DIC concentration of the algal medium was reduced from that in equilibrium with ambient air to even lower levels. This increased affinity was not correlated with any further increase in the CA_ex activity. Dextran-bound sulfonamide (DBS. 100 μM bound ligand) completely inhibited the activity of CA_ex in intact, low-DIC grown, wild-type cells, while photosynthesis at <2 μM CO₂(aq) proceeded at a far greater rate than could be maintained by CO₂ supplied from the spontaneous dehydration of HCO^?₃. DBS-inhibition of CA_ex, during the induction of the DIC-accumulating mechanism in previously high-DIC grown cells, only caused a 50% inhibition of photosynthesis at 10 μM CO₂(aq) after 1 h of low-DIC acclimation. It was also shown that 50 μM acetazolamide (AZ) inhibited photosynthesis at low DIC concentrations to a relatively higher degree than DBS, suggesting that AZ inhibited intracellular CA as well. Taken together, these results suggest that low-DIC grown cells of C. reinhardtii have the ability to transport HCO^?₃ across the plasma membrane in addition to the CA_ex-mediated, facilitated diffusion and/or transport of CO₂. It is also suggested that the relative importance of these two fluxes (CO₂ or HCO^?₃) is dependent on the growth and experimental conditions. Facilitated CO₂ uptake seems to be most prevalent, supported by HCO^?₃-transport under more or less extreme situations, such as a reduction of CO₂ to extremely low concentrations, leakage of CA_ex to the medium as in cultures of cell-wall-less mutant cells or when the activity of CA_ex has been artificially inhibited.