ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE IN CHLAMYDOMONAS REINHARDTII : Influence on Flagellar Function and Regeneration

Adenosine 3',5'-cyclic monophosphate (cAMP) influences both flagellar function and flagellar regeneration in Chlamydomonas reinhardtii. The methylxanthine, aminophylline, which can cause a tenfold increase in cAMP level in C. reinhardtii, inhibits flagellar movement and flagellar regeneration by wild-type cells, without inhibiting cell multiplication. Caffeine, a closely related inhibitor, also inhibits flagellar movement and regeneration, but it inhibits cell multiplication too. Regeneration by a mutant lacking the central pair of flagellar microtubules was found to be more sensitive than wild type to inhibition by caffeine and to be subject to synergistic inhibition by aminophylline plus dibutyryl cAMP. Regeneration by three out of seven mutants with different flagellar abnormalities was more sensitive than wild type to these inhibitors. We interpret these results to mean that cAMP affects a component of the flagellum directly or indirectly, and that the responsiveness of that component to cAMP is enhanced by mutations which affect the integrity of the flagellum. The component in question could be microtubule protein.     

Flagellar Development During the Cell Cycle in Chlamydomonas reinhardtii

Flagellar development during the asexual synchronous cell cycle of Chlamydomonas reinhardtii (11.32 aM) was studied by light microscopy. Cell walls of sporangia of different developmental status were dissolved using gamete lysin (g-lysin) enabling direct observation of flagellar development. Flagellar growth in progeny cells exhibits a linear kinetic with a growth rate of 28 nm/min at 30°C leading to a flagellar length of 7–7.5 μm in 4–4.5 h. After this time the flagellar growth rate drops to 2.8 nm/min (as in interphase). Both flagella of a single cell and all flagella within a sporangium grow out at the same time and with the same rate. Cycloheximide (10 μg/ml) completely blocks flagellar development. If cycloheximide is removed flagellar growth resumes at the normal rate with no lag-phase. Flagellar development during the cell cycle in C. reinhardtii differs considerably from the well-studied model system of flagellar regeneration following amputation in the same species.     

IMMUNOLOGICAL IDENTIFICATION OF THE ALTERNATIVE OXIDASE IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Using a monoclonal antibody to the alternative oxidase from voodoo lily, we provide evidence that the green alga Chlamydomonas reinhardtii Dang, possesses a protein that is immunologically related to the higher plant alternative oxidase. Mitochondria were isolated from a cell wall-less mutant strain (CW-15), and the presence of cyanide-resistant oxygen consumption was confirmed in these mitochondria. The voodoo lily antibody was used as a probe for immunoblotting of sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels of mitochondrial proteins of C. reinhardtii. The antibody reacted with a protein from C. reinhardtii with the same molecular mass (36 kDa) as the alternative oxidase from voodoo lily and tobacco mitochondria. These results suggest that cyanide-resistant respiration in C. reinhardtii is mediated by a higher plant-type alternative oxidase.     

Calmodulin Sensitivity of the Flagellar Membrane Adenylate Cyclase and Signaling of Motile Responses by cAMP in Gametes of Chlamydomonas reinhardtii

Cyclic AMP (cAMP) has been shown to be a primary signal of the agglutination-induced mating events of flagellar tip activation, cell wall loss, and mating structure activation in the unicellular alga Chlamydomonas reinhardtii (Pasquale and Goodenough, Cell Biol. 105 (1987), 2279–2293). The flagellar membrane adenylate cyclase of Chlamydomonas is here shown to be inhibited in vitro by EGTA, La³⁺, and trifluoperazine, and to be stimulated in the presence of calcium by incubation with exogenous calmodulin. Also, the motility of detergent-extracted models of Chlamydomonas is shown to be enhanced by cAMP. These observations suggest the hypothesis that the twitching motility characteristic of agglutinating Chlamydomonas gametes may be signaled by cAMP produced locally within the flagella by a calmodulin-sensitive adenylate cyclase.     

Ultrastructure of Chlamydomonas eugametos palmelloids induced by chloroplatinic acid treatment.

Palmelloids induced in the unicellular green alga Chlamydomonas eugametos by chloroplatinic acid treatment have been studied electron microscopically. Thin-sectioned specimens revealed the multilayer nature of the cell walls after second division within the palmelloid. Although synchrony in cell division is lost, to a certain degree, within the palmelloid, the cells themselves appeared normal, and the presence of normal flagellar structure was confirmed. The presence of the eyespot was observed at the optical level as well as in the freeze-etched specimens. The above results support the hypothesis that the palmelloid condition of Chlamydomonas eugametos induced by chloroplatinic acid is due to an abnormality in cell wall formation rather than flagellar malfunction or loss.     

Induction of DNA double-strand breaks by zeocin in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> and the role of increased DNA double-strand breaks rejoining in the formation of an adaptive response

This study aimed to test the potential of the radiomimetic chemical zeocin to induce DNA double-strand breaks (DSB) and “adaptive response” (AR) in Chlamydomonas reinhardtii strain CW15 as a model system. The AR was measured as cell survival using a micro-colony assay, and by changes in rejoining of DSB DNA. The level of induced DSB was measured by constant field gel electrophoresis based on incorporation of cells into agarose blocks before cell lysis. This avoids the risk of accidental induction of DSB during the manipulation procedures. Our results showed that zeocin could induce DSB in C. reinhardtii strain CW15 in a linear dose-response fashion up to 100 μg ml⁻¹ which marked the beginning of a plateau. The level of DSB induced by 100 μg ml⁻¹ zeocin was similar to that induced by 250 Gy of gamma-ray irradiation. It was also found that, similar to gamma rays, zeocin could induce AR measured as DSB in C. reinhardtii CW15 and this AR involved acceleration of the rate of DSB rejoining, too. To our knowledge, this is the first demonstration that zeocin could induce AR in some low eukaryotes such as C. reinhardtii.     

Identification of a Chlamydomonas plastidial 2‐lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content

Despite a strong interest in microalgal oil production, our understanding of the biosynthetic pathways that produce algal lipids and the genes involved in the biosynthetic processes remains incomplete. Here, we report that Chlamydomonas reinhardtii Cre09.g398289 encodes a plastid‐targeted 2‐lysophosphatidic acid acyltransferase (CrLPAAT1) that acylates the sn‐2 position of a 2‐lysophosphatidic acid to form phosphatidic acid, the first common precursor of membrane and storage lipids. In vitro enzyme assays showed that CrLPAAT1 prefers 16:0‐CoA to 18:1‐CoA as an acyl donor. Fluorescent protein‐tagged CrLPAAT1 was localized to the plastid membrane in C. reinhardtii cells. Furthermore, expression of CrLPAAT1 in plastids led to a > 20% increase in oil content under nitrogen‐deficient conditions. Taken together, these results demonstrate that CrLPAAT1 is an authentic plastid‐targeted LPAAT in C. reinhardtii, and that it may be used as a molecular tool to genetically increase oil content in microalgae.     

Isolation and Characterization of Chlamydomonas reinhardtii Mutants with Defects in the Induction of Flagellar Protein Synthesis after Deflagellation1,2

Amputating the flagella of Chlamydomonas reinhardtii stimulates increased synthesis of many flagellar proteins within 30 min. We have isolated a series of mutants which are defective in this stimulation, taking advantage of the fact that cells which cannot stimulate flagellar protein synthesis cannot regenerate flagella. More than a dozen mutants which have flagella, but cannot regenerate them after amputation, were isolated and studied by in vivo labeling to identify those non-regenerator mutants which were specifically defective in the induction of flagellar protein synthesis. Ten such mutants have been identified, and in each of them flagellar amputation does not stimulate the synthesis of any of the major flagellar proteins. At least four of the mutants display an interesting conditional phenotype. The synthesis of flagellar proteins after deflagellation is defective only in gametic cells; vegetative cells of these mutants are capable of flagellar protein synthesis after flagellar amputation.     

Cyclic AMP is one of the intracellular signals during the mating of Chlamydomonas eugametos

Chlamydomonas eugametos gametes of opposite mating type make cell-cell contact via their flagellar surfaces. This contact triggers an increase in the intracellular level of cyclic AMP (cAMP) and several cellular responses which are necessary for cell fusion. Here, we show that wheat-germ agglutinin, which binds to the flagellar surface and induces all mating responses, also increased the intracellular cAMP level. Dibutyryl-cAMP added to non-mating gametes induced flagellar twitching, cell-wall lysis, mating-structure activation, flagellartip activation and an increase in agglutinability. It did not induce agglutinin transport to the flagellar tip (tipping) and may not be the direct cause of flagellar twitching and flagellar-tip activation. In non-illuminated cells, dibutyryl-cAMP was far more effective in evoking mating reactions than in illuminated cells. Light induced a 50% decrease in the cAMP level within 1 min. Adenylate cyclase was found to be associated with cell membranes but only 8% of the total was present in the gamete flagella.Abbreviations db-cAMP dibutyryl-cAMP - FTA flagellar tip activation - Mab monoclonal antibody - mt ^–/mt⁺ mating-type minus/plus - WGA wheat-germ agglutinin We gratefully acknowledge the fruitful discussions with Dr. Rainer Gilles of the Department of Biochemistry at the University of Cologne (FRG), and the advice generously given by Dr. Roel van Driel of the Department of Biochemistry at the University of Amsterdam (The Netherlands).     

Altered Flagellar Size-Control in shf-1 Short-Flagella Mutants of Chlamydomonas reinhardtii

Two allelic Mendelian mutations which confer a short flagella phenotype were used to explore flagellar size control in Chlamydomonas reinhardtii. When mutant/wild type quadriflagellate dikaryon cells were constructed, their two short flagella rapidly grew out to near wild type length. The kinetics of elongation suggest that the flagellar assembly process is not intrinsically self-limiting as a number of otherwise attractive models for size control require. Instead, we suggest that there exists a cellular machinery dedicated to flagellar size control and that the short-flagella mutations alter the machinery in some as yet unknown way. One of the mutants shows temperature-sensitive flagellar assembly, and both are flagellaless in acetate media. Genetic analysis indicates that the temperaturesensitive, acetate-sensitive, and short-flagella phenotypes have a common genetic basis. The responsible gene has been named shf-1, and it has been mapped to chromosome VI, approximately 5 map units from the centromere.     

The Chlamydomonas chloroplast clpP gene contains translated large insertion sequences and is essential for cell growth

Sequence determination of the chloroplast clpP gene from two distantly related Chlamydomonas species (C. reinhardtii and C. eugametos) revealed the presence of translated large insertion sequences (IS1 and IS2) that divide the clpP gene into two or three sequence domains (SDs) and are not found in homologous genes in other organisms. These insertion sequences do not resemble RNA introns, and are not spliced out at the mRNA level. Instead, each insertion sequence forms a continuous open reading frame with its upstream and downstream sequence domains. IS1 specifies a potential polypeptide sequence of 286 and 318 amino acid residues in C. reinhardtii and C. eugametos, respectively. IS2 encodes a 456 amino acid polypeptide and is present only in C. eugametos. The two Chlamydomonas IS1 sequences show substantial similarity; however, there is no significant sequence similarity either between IS1 and IS2 or between these insertion sequences and any other known protein coding sequences. The C. reinhardtii clpP gene was further shown to be essential for cell growth, as demonstrated through targeted gene disruption by particle gun-mediated chloroplast transformation. Only heteroplasmic transformants could be obtained, even under mixotrophic growth conditions. The heteroplasmic transformants were stable only under selection pressure for the disrupted clpP, rapidly segregated into wild-type cells when the selection pressure was removed, and grew significantly more slowly than wildtype cells under phototrophic conditions.     

BIOSYNTHESIS OF POLY‐3‐HYDROXYBUTYRATE (PHB) IN THE TRANSGENIC GREEN ALGA CHLAMYDOMONAS REINHARDTII1

A cell‐wall deficient strain of Chlamydomonas reinhardtii P. A Dang. CC‐849 was cotransformed with two expression vectors, p105B124 and pH105C124, containing phbB and phbC genes, respectively, from Ralstonia eutropha. The transformants were selected on Tris‐acetate‐phosphate media containing 10 μg · mL^?1 Zeomycin. Upon further screening, the transgenic algae were subcloned and maintained in culture. PCR analysis demonstrated that both phbB and phbC genes were successfully integrated into the algal nuclear genome. Poly‐3‐hydroxybutyrate (PHB) synthase activity in these transgenic algae ranged from 5.4 nmol · min^?1 · mg protein^?1 to 126 nmol · min^?1 · mg protein^?1. The amount of PHB in double transgenic algae was determined by gas chromatography–mass spectrometry (GC–MS) when comparing with PHB standard. In addition, PHB granules were observed in the cytoplasm of transgenic algal cells using TEM, which indicated that PHB was synthesized in transgenic C. reinhardtii. Hence, results clearly showed that producing PHB in C. reinhardtii was feasible. Further studies would focus on enhancing PHB production in the transgenic algae and targeting the chloroplast for PHB accumulation.     

Selective Inhibition of Flagellar Activity in Chlamydomonas by Nickel

Flagellar activity in the biflagellate chlorophyte Chlamydomonas reinhardtii is selectively inhibited by Ni²⁺ or by treatment with Ca²⁺-chelating agents. Inhibitions of swimming speed, geotaxis, phototaxis, and pattern swimming result from qualitative and quantitative losses in the activity of individual flagella and in the coordination of activity beween the 2 flagella of each cell. Addition of Ca²⁺ (a) prevents inhibition and (b) restores normal flagellar activity in inhibited cells. Mg²⁺ is partially effective in reversal of inhibition. Other ions do not cause similar inhibition or reversal of nickel inhibition. The characteristics of inhibition and reversal suggest that the prmary target for nickel is a component of the flagellar apparatus, and that this component uses Ca²⁺ to perform its normal function in the regulation of flagellar activity. A 2nd target for nickel is a Carequiring process specific to phototaxis (and not involved in the photophobic response).     

IMMUNOLOCALIZATION OF CENTRIN IN OXYRRHIS MARINA (DINOPHYCEAE)1

A new polyclonal antibody was raised against centrin isolated from the flagellate green alga Spermatozopsis similis (Chlorophyta; anti-SSC). It stains by immunofluorescence and immunoelectron microscopy well-known reference systems for centrin like the nucleus–basal body connectors in Chlamydomonas reinhardtii (Chlorophyta) and the system II fibers (rhizoplasts) of Scherffelia dubia (Chlorophyta). In addition, it recognizes in immunoblots a single 20-kDa protein in isolated cytoskeletons of Spermatozopsis similis and Tetraselmis striata (Chlorophyta) as well as purified centrin isolated from Tetraselmis striata. Using this antibody, centrin was localized in whole cells and isolated cytoskeletons of Oxyrrhis marina Dujardin (Dinophyceae) by immunofluorescence and immunogold electron microscopy. In the flagellar apparatus of O. marina, five different structures were antigenic. Four short fibers (connectives 1–4) link the basal bodies to the four major fibrous flagellar roots, which do not cross-react with anti-centrin. The most prominent of the labeled structures (connective 5), a crescent-shaped fiber, extends from the flagellar canal of the transverse flagellum along the base of the tentacle to the flagellar canal of the longitudinal flagellum, interconnecting the distal parts of the microtubular roots/bands in the basal apparatus. For most of its length, it underlies and is connected to a transversely oriented subamphiesmal microtubular band. In immunoblot analyses, anti-SSC recognizes only a single 20-kDa protein in cytoskeletons of O. marina. Functional and phylogenetic aspects of centrin-containing structures in dinoflagellates are discussed.     

Temperature-Sensitive Chlamydomonas Mutants Manifesting Flagellar Regression at a Restrictive Temperature‡

The regeneration kinetics of Chlamydomonas reinhardtii mutants TS-6 and TS-79, whose flagella were mechanically amputated, indicated that the flagellar precursor in cytoplasm was used for regeneration when cycloheximide was present. The TS-6 cells rendered nonflagellate by regression at 35 C did not regenerate in the presence of cycloheximide, indicating that the precursor was inactivated by the high temperature. Neither mutant was able to use the absorbed flagellar components for regeneration in the presence of cycloheximide.     

Anomalies in the motion dynamics of long-flagella mutants of Chlamydomonas reinhardtii

Chlamydomonas reinhardtii has long been used as a model organism in studies of cell motility and flagellar dynamics. The motility of the well-conserved ‘9+2’ axoneme in its flagella remains a subject of immense curiosity. Using high-speed videography and morphological analyses, we have characterized long-flagella mutants (lf1, lf2-1, lf2-5, lf3-2, and lf4) of C. reinhardtii for biophysical parameters such as swimming velocities, waveforms, beat frequencies, and swimming trajectories. These mutants are aberrant in proteins involved in the regulation of flagellar length and bring about a phenotypic increase in this length. Our results reveal that the flagellar beat frequency and swimming velocity are negatively correlated with the length of the flagella. When compared to the wild-type, any increase in the flagellar length reduces both the swimming velocities (by 26–57%) and beat frequencies (by 8–16%). We demonstrate that with no apparent aberrations/ultrastructural deformities in the mutant axonemes, it is this increased length that has a critical role to play in the motion dynamics of C. reinhardtii cells, and, provided there are no significant changes in their flagellar proteome, any increase in this length compromises the swimming velocity either by reduction of the beat frequency or by an alteration in the waveform of the flagella.     

APOPTOTIC‐LIKE CELL DEATH PATHWAY IS INDUCED IN UNICELLULAR CHLOROPHYTE CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE) CELLS FOLLOWING UV IRRADIATION: DETECTION AND FUNCTIONAL ANALYSES1

Chlamydomonas reinhardtii (Ehrenberg) cells exhibited cell death process akin to that of apoptosis when exposed to ultraviolet (UV)‐C irradiation (1–100 J/m²). We observed typical hallmarks of apoptosis including cell shrinkage, associated nuclear morphological changes, flipping of phosphatidylserine, and DNA fragmentation detected by the terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling assay and oligonucleosomal DNA laddering assay. Interestingly, fluorescence imaging of DNA changes in UV‐C exposed cells, following PicoGreen staining, revealed that extra‐nuclear DNA disintegrates before that of nuclear changes, where the latter extensively diffuses out of the nuclear compartment, spreading into the whole cell and reaching the periphery of dying cells. Antibodies against a mammalian caspase‐3 shared epitopes with a protein of 28 kDa; whose pattern of expression correlated with the onset of cell death. Moreover, growth experiments indicate that spent medium recovered from UV‐C exposed cells exhibit a protective effect against cell killing of fresh cultures of C. reinhardtii cells by UV irradiation. The protective effect of UV‐spent medium is not a general growth promotional response on normal cells, but rather, is specific to UV‐exposed cells. We propose a model that C. reinhardtii cells exposed to UV elicit apoptotic‐like changes, which in turn lead to an adaptive response in neighboring cells against fresh rounds of UV exposure, thereby promoting survival of the cell population.     

The recombination and expression of the allophycocyanin beta subunit gene in the chloroplast of Chlamydomonas reinhardtii

Summary A Chlamydomonas reinhardtii (C. reinhardtii) chloroplast expression vector, papc-B, containing the apc-B gene that encodes the beta subunit of the light-harvesting antenna protein allophycocyanin (APC) of cyanobacteria, was constructed and transferred to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by Southern blot, Western blot and ELISA assays after selection on resistant medium. The recombinant APC beta subunit was expressed in the C. reinhardtii chloroplast and accounted for up to 2–3% (w/w) of the total soluble protein (TSP), suggesting a promising prospect of using C. reinhardtii chloroplasts to produce functional plant-derived proteins.     

Cyclic AMP functions as a primary sexual signal in gametes of Chlamydomonas reinhardtii

When Chlamydomonas reinhardtii gametes of opposite mating type are mixed together, they adhere by a flagella-mediated agglutination that triggers three rapid mating responses: flagellar tip activation, cell wall loss, and mating structure activation accompanied by actin polymerization. Here we show that a transient 10-fold elevation of intracellular cAMP levels is also triggered by sexual agglutination. We further show that gametes of a single mating type can be induced to undergo all three mating responses when presented with exogenous dibutyryl-cAMP (db-cAMP). These events are also induced by cyclic nucleotide phosphodiesterase inhibitors, which elevate endogenous cAMP levels and act synergistically with db-cAMP. Non-agglutinating mutants of opposite mating type will fuse efficiently in the presence of db-cAMP. No activation of mating events is induced by calcium plus ionophores, 8-bromo-cGMP, dibutyryl-cGMP, nigericin at alkaline pH, phorbol esters, or forskolin. H-8, an inhibitor of cyclic nucleotide-dependent protein kinase, inhibits mating events in agglutinating cells and antagonizes the effects of cAMP on non-agglutinating cells. Adenylate cyclase activity was detected in both the gamete cell body and flagella, with the highest specific activity displayed in flagellar membrane fractions. The flagellar membrane adenylate cyclase is preferentially stimulated by Mn++, unresponsive to NaF, GTP, GTP gamma S, AlF4-, and forskolin, and is inhibited by trifluoperazine. Cyclic nucleotide phosphodiesterase activity is also present in flagella. Our observations indicate that cAMP is a sufficient initial signal for all of the known mating reaction events in C. reinhardtii, and suggest that the flagellar cyclase and/or phosphodiesterase may be important loci of control for the agglutination-stimulated production of this signal.