Photosynthetic Properties of Chloroplasts from Chlamydomonas reinhardii

Chloroplasts isolated from synchronous cultures of the unicellular green alga Chlamydomonas reinhardii, SAG 11-32/b (−), fix CO₂ 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 CO₂ fixing capability per hour. Under reducing conditions (+ 1 millimolar dithiothreitol), this loss of activity is about twice as fast. The same reducing conditions stimulate CO₂ fixation in the light.

High concentrations of inorganic phosphate (>2 millimolar) inhibit CO₂ 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 Mg²⁺ concentration, the results were similar to that reported for chloroplasts from higher plants. However, in contrast to higher plant chloroplasts, maximum CO₂ fixation is observed at the relatively low osmotic concentration of 0.12 molar mannitol in the reaction buffer.

     

Energy Supply for ATP-Synthase Deficient Chloroplasts of Chlamydomonas reinhardii

The mutant F54 of the unicellular green alga Chlamydomonas reinhardiiis not able to perform photophos-phorylation. Nevertheless,it grows on acetate and the chloroplasts accomplish most oftheir energy-requiring synthetic processes. However, no light-dependentchloroplast protein synthesis could be detected in intact F54chloroplasts isolated from a cell wall-deficient double mutantF54-cw-15. Exogenous ATP was not able to induce this in organelloprotein synthesis to an appreciable degree. In contrast, thestrictly ATP-dependent protein synthesis was stimulated veryefficiently by glyceraldehyde-3-phosphate, dihydroxy-acetonephosphate and glycerol-3-phosphate, but strongly inhibited by3-phosphoglycerate. These compounds can be transported acrossthe 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, enhancedtriose phosphate-stimulated protein synthesis even further.In the dark, no stimulation was observed, indicating that alight-dependent reaction was also involved in this kind of ATPproduction in chloroplasts. The results suggest that chloroplastsdefective in photophosphorylation recruit their energy via anATP shuttle which was shown in this study to import rather thanexport ATP across the chloroplast envelope. (Received August 21, 1997; Accepted November 18, 1997)     

Biosynthesis of ferredoxin in Chlamydomonas reinhardii

The selective action of the antibiotics chloramphenicol and cycloheximide on the synthesis of ferredoxin in liquid cultures of Chlamydomonas reinhardii was studied. Highly specific antibodies raised against Chlamydomonas ferredoxin were used to determine the in vivo synthesis of apoferredoxin and conversion into native protein. The results indicate that 80S ribosomes are involved in the synthesis. Chlamydomonas cells growing in the absence of iron did not synthesize immunologically detectable amounts of ferredoxin. We suggest that this is based upon feed-back inhibition of apoferredoxin synthesis at the translational level.Abbreviations CAP chloramphenicol - CHI cycloheximide - IgG Immunoglobulin G - PBS 140.4 mM NaCl. 9 mM Na₂HPO₄, 1.3 mM NaH₂PO₄ (pH 74) - SDS sodium dodecvl sulphate - Fd Ferredoxin - apoFd Apoferredoxin - CM-Fd Scarboxymethylated Fd - TCA-Fd Fd treated with trichloroacetic acid     

Urate oxidase of Chlamydomonas reinhardii

Urate oxidase (EC 1.7.3.3) of Chlamydomonas reinhardii cells grown on purines and purine derivatives has been partially characterized. Crude enzyme preparations have a pH optimum of 9.0, require O₂ for activity, have an apparent K_m of 12 μ M for urate, and are inhibited by high concentrations of this substrate. Enzyme activity was particularly sensitive to metal ion chelating agents like cyanide, cupferron, diethyldithiocarbamate and o -phenanthroline, and to structural analogues of urate like hypoxanthine and xanthine. Chlamydomonas cells grow phototrophically on adenine, guanine, hypoxanthine, xanthine, urate, allantoin or allantoate as sole nitrogen source, indicating that in this alga the standard pathway of aerobic degradation of purines of higher plants, animals and many microorganisms operates. As deduced from experiments in vivo , urate oxidase from Chlamydomonas is repressed in the presence of ammonia or nitrate.     

Variability of mitochondrial population in Chlamydomonas reinhardii

Several details have been published cocerning the mitochondrial number and shapes at various stages of the synchronized vegetative and generative cell cycle in Chlamydomonas reinhardii. The present study, based on ultrathin serial sections and threedimensional reconstructions, completes these data. Quantitative analysis of serial micrographs makes it possible to give specific details of mitochondrial volumes in cells at early intermediate stages of the vegetative life cycle. Our investigations clearly show that mitochondria have a relatively wide range of sizes, within certain limits, and vary like the mitochondrial shapes; in fact, they vary in various cells at various stages as well as in several cells at the same stage and even in one and the same cell. Thus, we present a plastic insight into the dynamically changing micromorphology of the mitochondrial population in Chlamydomonas reinhardii.     

Localization of a non-Mendelian gene in Chlamydomonas reinhardii

Summary Transfer of a non-Mendelian neamine-dependent (nd) mutant to an antibioticfree medium results in neamine-sensitive and neamine-resistent revertants. These reversions are caused by extranuclear mutations.The neamine-sensitive revertants are no more able to split offnd-cells after back-donation to neamine containing medium. Therefore they are different from the streptomycin-sensitive revertants of a streptomycin-dependent (sd) mutant. These mutants were capable ofsd-segregation though their potence ofsd-segregation diminished on antibiotic-free medium with increasing time of cultivation.The different behaviour can be explained by the fact that manysd-genes are present which have to be appointed to the mitochondria. On the other side, thend-gene exists only in few copies and is located therefore in the chloroplast.Several experiments with differing methods are discussed to localize the extranuclear genes.

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Vorgelegt durch G. Melchers     

Degradation and compartmentalization of urea in Chlamydomonas reinhardii

Urea is accumulated against a concentration gradient in Chlamydomonas reinhardii. Only energy generated from photosynthesis is used for this accumulation, while degradation of urea utilizes other energy sources. Exogenous supplied urea is distributed between two pools, one large nonmetabolic and one metabolic pool. Ammonia inhibits the transport from the nonmetabolic to the metabolic pool.The nonmetabolic pool is probably located in the chloroplast, and the accumulation of urea is due to an active transport into the chloroplast.Non-Standard Abbreviations N medium medium without nitrogen source - TCA trichloroacetic acid - TCAI trichloroacetic acid insoluble fraction     

The nitrate-reducing enzyme system of Chlamydomonas reinhardii

In Chlamydomonas reinhardii the reduction of nitrate to ammonia occurs in two independent enzymatic steps: 1. the two-electrons reduction of nitrate to nitrite catalyzed by NADH-nitrate reductase, and, 2. the six-electrons reduction of nitrite to ammonia catalyzed by ferredoxin-nitrite reductase. Both enzymes have been purified and characterized, and some of their properties have been studied.     

The biological clock of Chlamydomonas reinhardii in space

The overt circadian rhythm in a wildtype (wt+) and a short period (s-) strain of Chlamydomonas reinhardii has been studied in space using the photoaccumulation behavior as the recorded parameter. The period of the wt+ was 29.6 h, of the s- 21.4 h and did not deviate significantly from ground controls performed exactly at the same time. The phase was delayed in space by 4.2 h in the wt+, but was not altered in the s-. In both strains the amplitudes were significantly higher in space than in the ground controls. During the recording period of 6.5 days the cell density increased in both strains. The survival rate, i.e. the ability to form colonies on agar petri dishes, was higher in space than on ground. The period was in both strains by 1.1 h longer in Florida (Kennedy Space Center) in both the flight and the control samples than in Europe. The significance of these results is discussed with respect to the endogenous nature of the biological clock and the role of the microgravity environment.     

Determination of cytochromes in chloroplasts of Chlamydomonas reinhardii mutants

A method for qualitative determination of cytochromes of the wild-type and mutant strains of Chalmydomonas reinhardii was developed. The effect of different techniques of cell disruption (ultrasound, Triton X-100, acetone, etc) on detection of cytochrome maxima in the oxidized minus reduced difference spectra was studied on a comparative basis. The development of the stable difference spectrum of the disrupted cell suspension was shown to be a function of incubation time in the presence of an oxidizing or a reducing agent. Cytochromes of the wild-type and 10 nonphotosynthesizing mutants were determined. Four mutants with lesions in cytochromes B559 and C553 were detected. Mutants with lesions in the reaction center of photosystem 2 were found to have a substantially reduced content of cytochrome B559, whereas those with normal photosystems--of cytochrome C553.     

Calcium lonophore-induced stimulation of secretory activity in Chlamydomonas reinhardii

The cell-wall lysin in gametes from Chlamydomonas reinhardii which under normal mating conditions is activated by flagellar cell contact was found to be susceptible to stimulation by the antibiotic ionophore A 23187 provided that CA²⁺ was included in the medium. Ionophore-induced release of the cell-wall lysin did not deend on the mating type or the gametic state of the cells. Vegetative cells which normally do not exhibit any mating capacity reacted with cell-wall lysis like gametes stimulated by cell contact.Ionophore-dependent Ca²⁺-transfer across the cell membranes generated a signal for cell-wall lysis only in cells with intact flagella. Deflagellated cells did not respond to A 23187 before regeneration of the amputated organelles. Another indication for a possible role of flagella in Ca²⁺-mediated cell-wall lysis was obtained from a conditional flagellar-assembly mutant of C. reinhardii which had been isolated and described by Huang et al. (1977). Upon shift-up the mutant strain immediately became unresponsive to A 23187 and Ca²⁺ but regained susceptibility soon after being retransferred to permissive conditions (20°C).     

A model of carbon dioxide assimilation in Chlamydomonas reinhardii

A simple model of photosynthetic CO₂ assimilation in Chlamydomonas has been developed in order to evaluate whether a CO₂-concentrating system could explain the photosynthetic characteristics of this alga (high apparent affinity for CO₂, low photorespiration, little O₂ inhibition of photosynthesis, and low CO₂ compensation concentration). Similarly, the model was developed to evaluate whether the proposed defects in the CO₂-concentrating system of two Chlamydomonas mutants were consistent with their observed photosynthetic characteristics. The model treats a Chlamydomonas cell as a single compartment with two carbon inputs: passive diffusion of CO₂, and active transport of HCO ₃ ^- . Internal inorganic carbon was considered to have two potential fates: assimilation to fixed carbon via ribulose 1,5-bisphosphate carboxylase-oxygenase or exiting the cell by either passive CO₂ diffusion or reversal of HCO ₃ ^- transport. Published values for kinetic parameters were used where possible. The model accurately reproduced the CO₂-response curves of photosynthesis for wild-type Chlamydomonas, the two mutants defective in the CO₂-concentrating system, and a double mutant constructed by crossing these two mutants. The model also predicts steady-state internal inorganic-carbon concentrations in reasonable agreement with measured values in all four cases. Carbon dioxide compensation concentrations for wild-type Chlamydomonas were accurately predicted by the model and those predicted for the mutants were in qualitative agreement with measured values. The model also allowed calculation of approximate energy costs of the CO₂-concentrating system. These calculations indicate that the system may be no more energy-costly than C₄ photosynthesis.Abbreviations Chl chlorophyll - RuBPC/O ribulose 1,5-bisphosphate carboxylase-oxygenase - CA carbonic anhydrase