Unicellular Green Alga Chlamydomonas Reinhardii Research Articles

Energy Supply for ATP-Synthase Deficient Chloroplasts of Chlamydomonas reinhardii

The mutant F54 of the unicellular green alga Chlamydomonas reinhardii is not able to perform photophosphorylation. Nevertheless, it grows on acetate and the chloroplasts accomplish most of their energy-requiring synthetic processes. However, no light-dependent chloroplast protein synthesis could be detected in intact F54 chloroplasts isolated from a cell wall-deficient double mutant F54-cw-15. Exogenous ATP was not able to induce this in organello protein synthesis to an appreciable degree. In contrast, the strictly ATP-dependent protein synthesis was stimulated very efficiently by glyceraldehyde-3-phosphate, dihydroxyacetone phosphate and glycerol-3-phosphate, but strongly inhibited by 3-phosphoglycerate. These compounds can be transported across the envelope membrane by the triose phosphate translocator. Pyridoxal phosphate, a specific inhibitor of the translocator, abolished the stimulation by triose phosphates. Spermidine, which activates initiation of translation in chloroplasts, enhanced triose phosphate-stimulated protein synthesis even further. In the dark, no stimulation was observed, indicating that a light-dependent reaction was also involved in this kind of ATP production in chloroplasts. The results suggest that chloroplasts defective in photophosphorylation recruit their energy via an ATP shuttle which was shown in this study to import rather than export ATP across the chloroplast envelope.

Purification and characterization of protein disulphide-isomerase from the unicellular green alga Chlamydomonas reinhardii. A 120 kDa dimer antigenically distinct from the vertebrate enzyme

Protein disulphide-isomerase (PDI) has been isolated from the unicellular green alga Chlamydomonas reinhardii and purified by (NH4)2SO4 precipitation, gel filtration and DEAE-Sephacel, hydroxyapatite and f.p.l.c. chromatography. The active algal enzyme is a 120 kDa dimer with a subunit molecular mass of 60 kDa when determined by SDS/PAGE. Although similar in size to the previously isolated vertebrate PDIs, the algal enzyme is antigenically distinct, polyclonal antibodies against the algal PDI showing no cross-reactivity with the vertebrate enzyme on immunoblots, and vice versa. The anti-(algal PDI) antiserum did not inhibit algal PDI activity, and C. reinhardii PDI could be immobilized on anti-PDI-Protein A-Sepharose in active form. In contrast with the situation in vertebrates, where PDI functions as a subunit of prolyl 4-hydroxylase, the C. reinhardii PDI is not associated with the algal prolyl 4-hydroxylase.

Polypeptide pattern of the insoluble wall component of Chlamydomonas reinhardii and its variation during the vegetative cell cycle

The cell wall of the unicellular green alga Chlamydomonas reinhardii consists of several salt-soluble glycoproteins and a framework of insoluble glycoproteins (K. Roberts, Phil. Trans. R. Soc. London B268 (1974) 129–146). This insoluble framework of the Chlamydomonas cell wall was completely solubilized by chemical deglycosylation with either trifluoromethanesulfonic acid or hydrogen fluoride in pyridine. Mild treatments with alkali or acid, however, did not destroy the insoluble wall component. Our findings suggest that the polypeptide subunits of the insoluble glycoprotein framework may be cross-linked through carbohydrate side chains. We demonstrate here that the pattern of polypeptides released from the insoluble wall component during chemical deglycosylation varies in a cell-cycle dependent manner. The insoluble wall component of all the analyzed cell cycle stages contained relatively constant amounts of polypeptides subunits with apparent molecular weights of >300, 150 and 64 kDa. Polypeptides with apparent molecular weights of 47 kDa and 22 kDa accumulated during cell growth, but were not present in the insoluble wall component of released zoospores. After apparent cytokinesis, increasing amounts of a 60-kDa and a 43-kDa polypeptide were detected. These polypeptides disappeared during liberation of zoospores.

The lithium-chloride-soluble cell-wall layers of Chlamydomonas reinhardii contain several immunologically related glycoproteins

Cell-wall glycoproteins of the unicellular green alga Chlamydomonas reinhardii have been purified from LiCl extracts of intact cells by gel exclusion chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies were raised against several polypeptide components isolated from the LiCl extracts. All these antibodies specifically reacted with the cell surface of formaldehyde-fixed cells. They showed cross-reactivity with the different antigens and were also reactive against some other polypeptides present in the LiCl extracts of intact wild-type cells as shown by double-diffusion assays and immunoblot analyses. These antigens were largely missing in LiCl extracts from the cell-wall-deficient mutant CW-15. The pattern of immunologically related cell-wall polypeptides of C. reinhardii varied during the vegetative cell cycle and was found to be also dependent on the growth conditions. Dot-immunobinding assays on chemically modified cell-wall glycoproteins demonstrated differences between the various antibodies with respect to their specificities. Differences were observed especially with respect to their reactivities against chemically deglycosylated cell-wall polypeptides. Chemical deglycosylation generally reduced the binding of the different antibodies indicating that all these antibodies recognize carbohydrate side chains. Only two of these antibody preparations, raised against cell-wall glycoproteins of relative molecular mass 35 and 150 kilodaltons, were found to be strongly reactive against deglycosylated cell-wall polypeptides. When these antibodies were saturated with cell-wall-derived glycopeptides in order to abolish the binding to carbohydrate side chains, they still recognized the same cell-wall polypeptides as did the untreated antibodies. These findings indicate that the cross-reactivity of the different cell-wall polypeptides with the antibodies is not exclusively the consequence of similar glycosylation patterns but is also the result of the presence of similar structures within the non-glycosylated stretches of the polypeptide backbones. Cell walls isolated from growing tobacco pollen tubes contained a single polypeptide component which showed crossreactivity with the antibodies to the cell-wall glycoproteins of C. reinhardii.

The Chlamydomonas cell cycle is regulated by a light/dark-responsive cell-cycle switch

Cultures of the unicellular green alga Chlamydomonas reinhardii can be synchronized by light/dark cycling not only under photoautotrophic but also under mixotrophic growth conditions. We observed that cultures synchronized in the presence of acetate continue to divide synchronously for one cell-cycle period when transferred to heterotrophic growth conditions. This finding enabled us to investigate the differential effects of light on cell growth and cell division. When cells were exposed to continuous light at the beginning of the growth period they entered the division phase earlier than dark-grown cells as a consequence of an increased growth rate. Illumination at the end of the growth period, however, caused a considerable delay in cell division and an extended growth period. The light-induced delay in cell division was also observed in the presence of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. This finding demonstrates that cell division is directly influenced by a light/dard-responsive cell-cycle switch rather than by light/dark-dependent changes in energy metabolism. The importance of this light/dark control to the regulation of the Chlamydomonas cell cycle was investigated in comparison with other control mechanisms (size control, time control). We found that the light/dard-responsive cell-cycle switch regulates the transition from G1-to S-phase. This control mechanism is effective in cells which have attained the commitment to at least one round of DNA replication and division but have not attained the maximal cell mass which initiates cell division in the light.

Intracellular calcium redistribution during mating in Chlamydomonas reinhardii

Gametes of the unicellular green alga Chlamydomonas reinhardii recognize and adhere to cells of the opposite mating type by flagellar contact. Adhesion between these specialized organelles signals a rapid series of mating events which result in gamete fusion. The sequence of morphological changes (flagellar tip activation, cell wall loss, and mating structure elongation), which occur as a consequence of the sexual signalling, have been characterized. The signalling mechanisms have, however, not been defined. Calcium is known to be involved during fertilization of animal species. Increased intracellular free calcium, which can be achieved either by calcium influx or by mobilization of ions from intracellular stores, has been observed during activation of both eggs and sperm. A recent report by Bloodgood & Levin that gametes of C. reinhardii preloaded with 45Ca showed a transient increase in Ca efflux following mating, suggests that intracellular Ca redistribution may also accompany mating in this algal species. We have used X-ray microanalysis to analyze the subcellular distribution of bound calcium during mating in Chlamydomonas reinhardii. X-ray maps reveal that calcium is sequestered in discrete granules within the gamete cell body prior to mating and that during activation and cell fusion, calcium is diffuse throughout the cell. This suggests the possibility that calcium serves as a second messenger in this species.

Changes in the shape of mitochondria following osmotic stress to the unicellular green alga Chlamydomonas reinhardii.

The effects of various stresses on mitochondrial activity and structure within the unicellular green alga Chlamydomonas reinhardii were investigated using the fluorescent probe rhodamine 123. Within control cells, treatment with rhodamine 123 stained an intense fluorescent network, which was considered to be mitochondrial from the similarity in structure to models of mitochondria reconstructed from serial-section electron microscopy, and because this pattern of staining was abolished following the addition of metabolic inhibitors. Following osmotic shrinkage and rehydration, fragmentation of the mitochondrial network was observed within potentially viable cells. This was reversible within 1 h of resuspension in isotonic medium. Exposure of cells to hypertonic solutions of rapidly permeating compounds did not induce similar structural alterations. These changes in the mitochondria were confirmed by thin-section electron microscopy. In the presence of higher osmolalities of non-permeating compounds, which induce a greater loss of viability, rhodamine 123 stained cells uniformly. Following the osmotic stresses induced by slow rates of freezing and subsequent thawing no fragmentation in mitochondrial staining was observed. These findings demonstrate that shrinkage and rehydration may induce alterations to the structure and function of organelles and may be factors in determining cellular viability following osmotic stress.

Glycoprotein biosynthesis in Chlamydomonas. A mannolipid intermediate with the properties of a short-chain alpha-saturated polyprenyl monophosphate.

A crude membrane preparation of the unicellular green alga Chlamydomonas reinhardii was found to catalyse the incorporation of D-[14C]mannose from GDP-D-[14C]-mannose into a chloroform/methanol-soluble compound and into a trichloroacetic acid-insoluble polymer fraction. The labelled lipid revealed the chemical and chromatographic properties of a short-chain (about C55-C65) alpha-saturated polyprenyl mannosyl monophosphate. In the presence of detergent both long-chain (C85-C105) dolichol phosphate and alpha-unsaturated undecaprenyl phosphate (C55) were found to be effective as exogenous acceptors of D-mannose from GDP-D-[14C]mannose to yield their corresponding labelled polyprenyl mannosyl phosphates. Exogenous dolichyl phosphate stimulated the incorporation of mannose from GDP-D-[14C]mannose into the polymer fraction 5-7-fold, whereas the mannose moiety from undecaprenyl mannosyl phosphate was not further transferred. Authentic dolichyl phosphate [3H]mannose and partially purified mannolipid formed from GDP-[14C]mannose and exogenous dolichyl phosphate were found to function as direct mannosyl donors for the synthesis of labelled mannoproteins. These results clearly indicate the existence of dolichol-type glycolipids and their role as intermediates in transglycosylation reactions of this algal system. Both the saturation of the alpha-isoprene unit and the length of the polyprenyl chain may be regarded as evolutionary markers.

Reduced Inorganic Carbon Transport in a CO2-Requiring Mutant of Chlamydomonas reinhardii

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated that is deficient in inorganic carbon transport. This mutant strain, designated pmp-1-16-5K (gene locus pmp-1), was selected on the basis of a requirement of elevated CO(2) concentration for photoautrophic growth. Inorganic carbon accumulation in the mutant was considerably reduced in comparison to wild type, and the CO(2) response of photosynthesis indicated a reduced affinity for CO(2) in the mutant. At air levels of CO(2) (0.03-0.04%), O(2) inhibited photosynthesis and stimulated the synthesis of photorespiratory intermediates in the mutant but not in wild type. Neither strain was significantly affected by O(2) at saturating CO(2) concentration. Thus, the primary consequence of inorganic carbon transport deficiency in the mutant was a much lower internal CO(2) concentration compared to wild type. From these observations, we conclude that enzyme-mediated transport of inorganic carbon is an essential component of the CO(2) concentrating system in C. reinhardii photosynthesis.

Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated which is deficient in carbonic anhydrase (EC 4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locus ca-1), was selected on the basis of a high CO(2) requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric CO(2) concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by O(2). In contrast to a CO(2) compensation concentration of near zero in wild type at all O(2) concentrations examined, the mutant exhibited a high, O(2)-stimulated CO(2) compensation concentration. Evidence of photorespiratory activity in the mutant but not in wild type was obtained from the analysis of photosynthetic products in the presence of (14)CO(2). At air levels of CO(2) and O(2), the mutant synthesized large amounts of glycolate, while little glycolate was synthesized by wild type under identical conditions. Both mutant and wild type strains formed only small amounts of glycolate at saturating CO(2) concentration. At ambient CO(2), wild type accumulated inorganic carbon to a concentration several-fold higher than that in the suspension medium. The mutant cells accumulated inorganic carbon internally to a concentration 6-fold greater than found in wild type, yet photosynthesis was CO(2) limited. The mutant phenotype was mimicked by wild type cells treated with ethoxyzolamide, an inhibitor of carbonic anhydrase activity. These observations indicate a requirement for carbonic anhydrase-catalyzed dehydration of bicarbonate in maintaining high internal CO(2) concentrations and high photosynthesis rates. Thus, in wild type cells, carbonic anhydrase rapidly converts the bicarbonate taken up to CO(2), creating a high internal CO(2) concentration which stimulates photosynthesis and suppresses photorespiration. In mutant cells, bicarbonate is taken up rapidly but, because of a carbonic anhydrase deficiency, is not dehydrated at a rate sufficiently rapid to maintain a high internal CO(2) concentration.

Photosynthetic Properties of Chloroplasts from Chlamydomonas reinhardii

Chloroplasts isolated from synchronous cultures of the unicellular green alga Chlamydomonas reinhardii, SAG 11-32/b (-), fix CO(2) at rates between 25 and 50 micromoles per milligram chlorophyll per hour. The upper value is approximately half of the rate of the intact cell.During storage in the dark on ice, the chloroplast preparation loses 30 to 50% of its CO(2) fixing capability per hour. Under reducing conditions (+ 1 millimolar dithiothreitol), this loss of activity is about twice as fast. The same reducing conditions stimulate CO(2) fixation in the light.High concentrations of inorganic phosphate (>2 millimolar) inhibit CO(2) fixation. This inhibition is overcome by the addition of glycerate 3-phosphate. It is concluded that chloroplasts from C. reinhardii possess a higher plant type phosphate translocator. With respect to dependency upon light intensity, pH and Mg(2+) concentration, the results were similar to that reported for chloroplasts from higher plants. However, in contrast to higher plant chloroplasts, maximum CO(2) fixation is observed at the relatively low osmotic concentration of 0.12 molar mannitol in the reaction buffer.

Purification and properties of carbonic anhydrase from Chlamydomonas reinhardii

Carbonic anhydrase (CA) was purified from the unicellular green alga Chlamydomonas reinhardii, and the purity of the preparation was established by gradient gel electrophoresis. The purified enzyme exhibited a MW of 165 000 and contained 6 atoms of Zn. The subunit MW, as determined by dodecyl sulfate electrophoresis, was 27 000. These results are consistent with a quarternary structure which is hexameric, each monomer containing 1 g atom of Zn. Like spinach CA, and in contrast to other oligomeric plant CAs, a sulfhydryl reducing agent is not needed to stabilize the enzyme. CO 2-hydrase activity was inhibited by both acetazolamide ( I 50 = 7.8 × 10 −9M) and sulfanilamide ( I 50 = 1.3 × 10 −5M), as well as by certain inorganic anions. The purified enzyme showed relatively weak esterase activity with p-nitrophenyl acetate but was an extremely effective esterase with 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone as the substrate. Both esterase activities could be completely inhibited by adding acetazolamide. In its gross structural characteristics, the C. reinhardii enzyme resembles the CAs from higher plants. However, in its esterase activity and the inhibition by sulfonamides it is markedly different from plant CAs and bears more resemblance to erythrocyte CAs.

Basal body and flagellar development during the vegetative cell cycle and the sexual cycle of Chlamydomonas reinhardii.

ABSTRACT Basal body development and flagellar regression and growth in the unicellular green alga Chlamydomonas reinhardii were studied by light and electron microscopy during the vegetative cell cycle in synchronous cultures and during the sexual life cycle. Flagella regress by gradual shortening prior to vegetative cell division and also a few hours after cell fusion in the sexual cycle. In vegetative cells basal bodies remain attached to the plasma membrane by their transitional fibres and do not act as centrioles at the spindle poles during division. In zygotes the basal bodies and associated microtubular roots and cross-striated connexions all dissolve, and by 6·5 h after mating all traces of flagellar apparatus and associated structures have disappeared. They remain absent for 6 days throughout zygospore maturation and then are reassembled during zygospore germination, after meiosis has begun. Basal body assembly in developing zygospores occurs close to the plasma membrane (in the absence of pre-existing basal bodies) via an intermediate stage consisting of nine single A-tubules surrounding a central ‘cartwheel’. Assembly is similar in vegetative cells (and occurs prior to cell division), except that new basal bodies are physically attached to old ones by amorphous material. In vegetative cells, amorphous disks, which may possibly be still earlier stages in basal-body development occur in the same location as 9-singlet developing basal bodies. After the 9-singlet structure is formed, B and C fibres are added and the basal body elongates to its mature length. Microtubular roots, striated connexions and flagella are then assembled. Both flagellar regression and growth are gradual and sequential, the transitional region at the base of the flagellum being formed first and broken down last. The presence of amorphous material at the tip of the axoneme of growing and regressing flagella suggests that the axoneme grows or shortens by the sequential assembly or disassembly at its tip. In homogenized cells basal bodies remain firmly attached to each other by their striated connexions. The flagellar transitional region, and parts of the membrane and of the 4 micro tubular roots, also remain attached; so also do new developing basal bodies, if present. These structures are well preserved in homogenates and new fine-structural details can be seen. These results are discussed, and lend no support to the idea that basal bodies have genetic continuity. It is suggested that basal body development can be best understood if a distinction is made between the information needed to specify the structure of a basal body and that needed to specify its location and orientation.

The aldolases of chlamydomonas reinhardii

The unicellular green alga Chlamydomonas reinhardii contains two distinct aldolases (fructose-1, 6-diphosphate D -glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13). These two enzymes are synthesized under different growth conditions: the alga can contain aldolase I or aldolase II or both of them. The two proteins are localized in the cytoplasm and they have been purified and compared, using kinetic and physical parameters. One resembles the prototype rabbit muscle aldolase, the other is a metalrequiring enzyme.