Induction of Inorganic Carbon Accumulation in the Unicellular Green Algae Scenedesmus obliquus and Chlamydomonas reinhardtii

The induction of a dissolved inorganic carbon (DIC) accumulating mechanism in the two algal species Scenedesmus obliquus (WT) and Chlamydomonas reinhardtii (137 c+) was physiologically characterized by monitoring DIC uptake kinetics at a low and constant DIC concentration (120-140 micromolar), after transfer from high-DIC culturing conditions. A potentiometric titration method was used to measure and calculate algal DIC uptake. Full acclimation to low-DIC conditions was obtained within a period of 90 min, after which time the DIC uptake had been increased 7 to 10 times. Experiments were also conducted in the presence of inhibitors against DIC accumulation. The inhibitor of extracellular carbonic anhydrase (CA), acetazolamide (50 micromolar), inhibited the adaptation partly, while the inhibitor of both extra- and intracellular CA, ethoxyzolamide (50 micromolar) totally inhibited the acclimation. Cycloheximide (10 micrograms per milliliter), which inhibits protein synthesis on cytoplasmic ribosomes, and vanadate (180 micromolar), which inhibits the plasmamembrane bound ATPase, also inhibited the acclimation totally. These results taken together suggest that the algae are dependent on intracellular CA, plasmamembrane bound ATPase, and de novo protein synthesis for DIC accumulation. Also, these components are more important than extracellular CA for the overall function of the DIC-accumulating mechanism.     

莱茵衣藻和斜生栅藻蛋白核的染色方法优化

蛋白核（pyrenoid）作为真核藻类重要的、相对独立的固碳结构,在CO2高效浓缩和固定过程中发挥中重要作用,可为开发高效生物固碳技术提供理论支持。染色法是快速观察蛋白核的最佳方法之一,有助于深入研究蛋白核的结构和功能,但目前对染色法仍缺乏系统研究。本文以较易染色的莱茵衣藻（Chlamydomonas reinhardtii）和较难染色的斜生栅藻（Scenedesmus obliquus）为材料,探讨了碘染和溴酚蓝（BPB）染色观察蛋白核的方法。结果表明,碘染法最适用于蛋白核外周淀粉观察,最适染色液浓度和时间分别是0．1％～0．2％（w／v）和5min。热激预处理和溴酚蓝染色相结合的蛋白核染色效果更好,优化条件为：70℃水浴热激40s,0．05％（w／v）BPB染色15min。本方法为研究微藻蛋白核提供了一种快速有效的直接观察方法。     

Low molecular weight subunits associated with the cytochrome b 6 f complexes from spinach and Chlamydomonas reinhardtii

Cytochrome b ₆ f complexes, prepared from spinach and Chlamydomonas thylakoids, have been examined for their content of low molecular weight subunits. The spinach complex contains two prominent low molecular weight subunits of 3.7 and 4.1 kD while a single prominent component of 4.5 kD was present in the Chlamydomonas complex. An estimation of the relative stoichiometry of these subunits suggests several are present at levels approximating one copy per cytochrome complex. The low molecular weight subunits were purified by reversed phase HPLC and N-terminal sequences obtained. Both the spinach and Chlamydomonas cytochrome complexes contain a subunit that is identified as the previously characterized petG gene product (4.8 kD in spinach and 4.1 kD in Chlamydomonas). A second subunit (3.8 kD in spinach and 3.7 kD in Chlamydomonas) appears to be homologous in the two complexes and is likely to be a nuclear gene product. The possible presence of other low molecular weight subunits in these complexes is also considered.     

Influence of mixing and shear stress on Chlorella vulgaris, Scenedesmus obliquus, and Chlamydomonas reinhardtii

Photosynthetic activity (PA) and growth of different microalgae species (Chlorella vulgaris, Scenedesmus obliquus, and Chlamydomonas reinhardtii) depends in addition to other factors on mixing (tip speed) and shear stress (friction velocity) and was studied in a stirring tank (microcosm). In order to detect cause–effect relationships for an increase in photosynthetic activity, experiments were conducted under different pH values (6.0–8.5) and CO₂ concentrations (0.038 and 4 % (v/v)). The PA was determined as the effective quantum yield by pulse amplitude modulation during a stepwise increase of the tip speed from 0 to 589 cm s^?1 (friction velocity: 0–6.05 cm s^?1) in short-term experiments. The increase caused a distinctive pattern of PA of each species. Compared to 0 cm s^?1, C. vulgaris and S. obliquus showed a 4.0 and 4.8 % higher PA at the optimum tip speed of 126 cm s^?1 (friction velocity of 2.09 cm s^?1) and a 48 and 71 % higher growth, respectively. At 203 cm s^?1, the PA dropped to the value of the unstirred control, while at 589 cm s^?1, the PA decreased of up to 7 and 8 %. In contrast, C. reinhardtii showed 7 % stronger growth at 126 cm s^?1, while the PA decreased about 15 % at an increase of tip speed to 589 cm s^?1. For all investigated microalgae, the pattern of PA and higher growth was not only explained by the main contributing factors like light supply, nutrient supply, and overcoming diffusion gradients. The results indicate that hydrodynamic forces have a stimulating effect on the physiological processes within the cells.     

A cytochrome b5-containing plastid-located fatty acid desaturase from Chlamydomonas reinhardtii

Monogalactosyldiacylglycerol (MGDG) in Chlamydomonas reinhardtii and other green algae contains hexadeca-4,7,10,13-tetraenoic acid (16:4) in the glycerol sn-2 position. While many genes necessary for the introduction of acyl chain double bonds have been functionally characterized, the Δ4-desaturase remained unknown. Using a phylogenetic comparison, a candidate gene encoding the MGDG-specific Δ4-desaturase from Chlamydomonas (CrΔ4FAD) was identified. CrΔ4FAD shows all characteristic features of a membrane-bound desaturase, including three histidine boxes and a transit peptide for chloroplast targeting. But it also has an N-terminal cytochrome b(5) domain, distinguishing it from other known plastid desaturases. Cytochrome b(5) is the primary electron donor for endoplasmic reticulum (ER) desaturases and is often fused to the desaturase domain in desaturases modifying the carboxyl end of the acyl group. Difference absorbance spectra of the recombinant cytochrome b(5) domain of CrΔ4FAD showed that it is functional in vitro. Green fluorescent protein fusions of CrΔ4FAD localized to the plastid envelope in Chlamydomonas. Interestingly, overproduction of CrΔ4FAD in Chlamydomonas not only increased levels of 16:4 acyl groups in cell extracts but specifically increased the total amount of MGDG. Vice versa, the amount of MGDG was lowered in lines with reduced levels of CrΔ4FAD. These data suggest a link between MGDG molecular species composition and galactolipid abundance in the alga, as well as a specific function for this fatty acid in MGDG.     

The assembly of cytochrome b6/f complexes: an approach using genetic transformation of the green alga Chlamydomonas reinhardtii.

As an approach to the study of the biogenesis of the cytochrome b6/f complex, we characterized the behaviour of its constitutive subunits in mutant strains of Chlamydomonas reinhardtii bearing well-defined mutations. To this end, we have constructed three deletion mutant strains, each lacking one of the major chloroplast pet genes: the delta petA, delta petB and delta petD strains were unable to synthesize cyt f, cyt b6 and subunit IV (suIV) respectively. Western blotting analysis, pulse-labelling and pulse-chase experiments allowed us to compare the cellular accumulation, the rates of synthesis and the turnover of the cyt b6/f subunits remaining in the various strains. We show that the rates of synthesis of cyt b6 and suIV are independent of the presence of the other subunits of the complex but that their stabilization in the thylakoid membranes is a concerted process, with a marked dependence of suIV stability on the presence of cyt b6. In contrast, mature cyt f was stable in the absence of either suIV or cyt b6 but its rate of synthesis was severely decreased in these conditions. We conclude that the stoichiometric accumulation of the chloroplast-encoded subunits of the cyt b6/f complex results from two regulation processes: a post-translational regulation leading to the proteolytic disposal of unassembled cyt b6 and suIV and a co-translational (or early post-translational) regulation which ensures the production of cyt f next to its site of assembly.     

The basal bodies of Chlamydomonas reinhardtii. Formation from probasal bodies, isolation, and partial characterization

The assembly and composition of basal bodies was investigated in the single-celled, biflagellate green alga, Chlamydomonas reinhardtii, using the cell wall-less strain, cw15. In the presence of EDTA, both flagellar axonemes remained attached to their basal bodies while the entire basal body-axoneme complex was separated from the cell body, without cell lysis, by treatment with polyethylene glycol-400. The axonemes were then removed from the basal bodies in the absence of EDTA, leaving intact basal body pairs, free from particulate contamination from other regions of the cell. The isolated organelles produced several bands on sodium dodecyl sulfate-urea polyacrylamide gels, including two tubilin bands which co-electrophoresed with flagellar tubulin. The formation of probasal bodies was observed by electron microscopy of whole mount preparations. Synchronous cells were lysed, centrifuged onto carbon-coated grids, and either negatively stained or shadowed with platinum. The two probasal bodies of each cell appeared shortly after mitosis as thin "annuli," not visible in thin sections, each consisting of nine rudimentary triplet microtubules. Each annulus remained attached to one of the mature basal bodies by several filaments about 60 in diameter, and persisted throughout interphase until just before the next cell division. It then elongated into a mature organelle. The results revive the possibility of the nucleated assembly of basal bodies.     

Potassium transport in Chlamydomonas reinhardtii: isolation and characterization of transport-deficient mutant strains

Putative potassium-transport-deficient mutant strains of Chlamydomonas reinhardtii Dang. were induced by ultra-violet mutagenesis and were identified by their dependence on abnormally high concentrations of potassium for growth. Potassium transport studies employing ⁸⁶Rb as a tracer were carried out with wild-type cells and with three independently isolated KDP (potassium-dependent phenotype) clones. Wildtype cells exhibit two transport activities. Transport activity A was expressed when cells were grown in medium supplemented with 10 mM KCl. The transporter with type-A activity does not discriminate between either Rb⁺ or K⁺ as a substrate and has a K_m for Rb⁺ equal to 1 mM and a V_max equal to 31 nmol Rb⁺ h^-1 10^-6 cells. Transport activity B was expressed when cells were starved of potassium for 24 h. The transporter with type-B activity prefers K⁺ to Rb⁺ as a substrate; it has a K_m for Rb⁺ equal to 2.5 mM and a V_max equal to 210 nmol Rb⁺ h^-1 10^-6 cells. All three mutant clones exhibit transport activity comparable to type-A when grown in 10 mM KCl. When starved of potassium for 24 h, two KDP clones demonstrate no transport activity and the third clone continues to exhibit only type-A activity.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DES diethylstilbesterol - KDP potassium-dependent phenotype     

Brownian dynamics study of cytochrome f interactions with cytochrome c6 and plastocyanin in Chlamydomonas reinhardtii plastocyanin, and cytochrome c6 mutants

Using Brownian dynamics simulations, all of the charged residues in Chlamydomonas reinhardtii cytochrome c(6) (cyt c(6)) and plastocyanin (PC) were mutated to alanine and their interactions with cytochrome f (cyt f) were modeled. Systematic mutation of charged residues on both PC and cyt c(6) confirmed that electrostatic interactions (at least in vitro) play an important role in bringing these proteins sufficiently close to cyt f to allow hydrophobic and van der Waals interactions to form the final electron transfer-active complex. The charged residue mutants on PC and cyt c(6) displayed similar inhibition classes. Our results indicate a difference between the two acidic clusters on PC. Mutations D44A and E43A of the lower cluster showed greater inhibition than do any of the mutations of the upper cluster residues. Replacement of acidic residues on cyt c(6) that correspond to the PC's lower cluster, particularly E70 and E69, was observed to be more inhibitory than those corresponding to the upper cluster. In PC residues D42, E43, D44, D53, D59, D61, and E85, and in cyt c(6) residues D2, E54, K57, D65, R66, E70, E71, and the heme had significant electrostatic contacts with cyt f charged residues. PC and cyt c(6) showed different binding sites and orientations on cyt f. As there are no experimental cyt c(6) mutation data available for algae, our results could serve as a good guide for future experimental work on this protein. The comparison between computational values and the available experimental data (for PC-cyt f interactions) showed overall good agreement, which supports the predictive power of Brownian dynamics simulations in mutagenesis studies.     

Cytoplasmic ribosomal proteins from Chlamydomonas reinhardtii: characterization and immunological comparisons

Summary Experiments were undertaken to characterize the cytoplasmic ribosomal proteins (r-proteins) in Chlamydomonas reinhardtii and to compare immunologically several cytoplasmic r-proteins with those of chloroplast ribosomes of this alga, Escherichia coli, and yeast. The large and small subunits of the C. reinhardtii cytoplasmic ribosomes were shown to contain, respectively, 48 and 45 r-proteins, with apparent molecular weights of 12,000–59,000. No cross-reactivity was seen between antisera made against cytoplasmic r-proteins of Chlamydomonas and chloroplast r-proteins, except in one case where an antiserum made against a large subunit r-protein cross-reacted with an r-protein of the small subunit of the chloroplast ribosome. Antisera made against one out of five small subunit r-proteins and three large subunit r-proteins recognized r-proteins from the yeast large subunit. Each of the yeast r-proteins has been previously identified as an rRNA binding protein. The antiserum to one large subunit r-protein cross-reacted with specific large subunit r-proteins from yeast and E. coli.     

Isolation and characterization of xanthine dehydrogenase from Chlamydomonas reinhardtii

Xanthine dehydrogenase (XDH, EC 1.2.1.37) of Chlamydomonas reinhardtii (Sager) 6145c wild strain has been isolated and characterized for the first time in a unicellular green alga. The enzyme has an M_r of 330 kDa, and FAD, molybdenum and iron are cofactors required for its activity as deduced from results obtained using specific inhibitors, ⁵⁹Fe-labelling experiments, activity protection by FAD, physiological responses in vivo to iron and molybdenum deficiencies in the culture medium and work with mutants lacking molybdenum cofactor. Xanthine dehydrogenase exhibited Mi-chaelian kinetics typical for a bisubstrate enzyme with apparent K_m values for NAD ⁺, hypoxanthine and xanthine of 35, 160 and 70 μ M , respectively. Under phototrophic conditions enzyme activity was repressed by ammonium, but xanthine was not required for the enzyme to be induced, since high levels of enzyme activity were found in cells grown on ammonium and transferred to either N-frec media or media containing either of the nitrogen sources adenine, urea, urate, xanthine, hypoxanthine and guanine.     

Lichen-like association of Chlamydomonas reinhardtii and Aspergillus nidulans protects algal cells from bacteria

Organismal interactions within microbial consortia and their responses to harmful intruders remain largely understudied. An important step toward the goal of understanding functional ecological interactions and their evolutionary selection is the study of increasingly complex microbial interaction systems. Here, we discovered a tripartite biosystem consisting of the fungus Aspergillus nidulans, the unicellular green alga Chlamydomonas reinhardtii, and the algicidal bacterium Streptomyces iranensis. Genetic analyses and MALDI-IMS demonstrate that the bacterium secretes the algicidal compound azalomycin F upon contact with C. reinhardtii. In co-culture, A. nidulans attracts the motile alga C. reinhardtii, which becomes embedded and surrounded by fungal mycelium and is shielded from the algicide. The filamentous fungus Sordaria macrospora was susceptible to azalomycin F and failed to protect C. reinhardtii despite chemotactically attracting the alga. Because S. macrospora was susceptible to azalomycin F, this data imply that for protection the fungus needs to be resistant. Formation of the lichen-like association between C. reinhardtii and A. nidulans increased algal growth. The protection depends on the increased amounts of membrane lipids provided by resistant fungi, thereby generating a protective shelter against the bacterial toxin. Our findings reveal a strategy whereby algae survive lethal environmental algicides through cooperation with fungi.Subject terms: Microbial ecology, Microbiome, Microbial ecology, Antibiotics, Fungal ecology     

Luminescence decay kinetics in relation to quenching and stimulation of dark fluorescence from high and low CO2 adapted cells of Scenedesmus obliquus and Chlamydomonas reinhardtii

Two green algal species, Chlamydomonas reinhardtii and Scenedesmus obliquus, exhibited a relative maximum during the decay of luminescence, when adapted to low CO₂ conditions that was not observed in high CO₂ adapted cells.From the kinetics of transient changes in the level of dark fluorescence, after illumination and parallel to the luminescence maxima, it was concluded that the maximum in Scenedesmus was mainly related to a decrease in nonphotochemical quenching, whereas in Chlamydomonas the maximum was mainly related to a dark reduction of the primary PS II acceptor Q_A.ATP/ADP ratios from low CO₂ adapted Scenedesmus showed transient high levels after a dark/light transition that was not observed in high CO₂ adapted cells. After 30 s of illumination the ATP/ADP ratios however stabilized at the same steady state level as in high CO₂ adapted cells.Dark addition of HCO₃ ^- to low CO₂ adapted cells of Chlamydomonas resulted in a rapid transient quenching of luminescence that was not observed in low CO₂ adapted cells of neither species.It is concluded that the luminescence maxima present in both low CO₂ adapted Scenedesmus and Chlamydomonas reflect adaptation of the cells to low CO₂ conditions. It is further suggested that the difference in mechanistic origin of luminescence maxima in the two species reflects differences in adaptation.Abbreviations ADP adenosine-diphosphate - ATP adenosine-triphosphate - C_i inorganic carbon - F_D dark fluorescence recorded under dark adapted conditions - F₀ fluorescence with all reaction centers open - FV variable fluorescence - PS I photosystem I - PS II photosystem II - Q_A the first quinone acceptor of PS II     

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

Cytochrome c6from 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 c6crystallized as a monomer in the space group P 21212, whereas the oxidized protein crystallized as a dimer in the space group P 3121. The structures were solved by molecular replacement and refined to 1. 9 and 2.0 A, 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 c6as observed in the oxidized crystal, is discussed with respect to its biological relevance and its implications for the reaction mechanisms between cytochrome c6and its redox partners. The dimer of oxidized cytochrome c6may represent a molecular structure occurring in a binary complex with cytochrome b6f. This assembly might be required for the correct orientation of cytochrome c6with respect to its redox partner cytochrome b6f, 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 c6, i.e. a random collision model for the formation of an electron transfer complex must be assumed.     

Purification and characterization of chloroplastic NADP-isocitrate dehydrogenase from Chlamydomonas reinhardtii

Chloroplastic NADP-isocitrate dehydrogenase isoenzyme (NADP-IDH₂; EC 1.1.1.42) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by a procedure which included affinity chromatography on Red-Sepharose as the key step. The 70-kDa isoenzyme was found to be a dimer formed by 40-kDa subunits. Antibodies raised against a recombinant tobacco cytosolic NADP-IDH cross-reacted strongly with the cytosolic NADP-IDH₁ and weakly with the NADP-IDH₂ isoenzyme from this alga. NADPH and GTP were found to inhibit both isoenzymes, whereas intermediates of the tricarboxylic acid cycle, glycolysis or reductive pentose phosphate cycle had no significant effect. The simultaneous presence of isocitrate and Mn²⁺ protected NADP-IDH₂ against thermal inactivation or inhibition by reagents specific for arginine or lysine.     

Oil extraction from Scenedesmus obliquus using a continuous microwave system--design, optimization, and quality characterization

A 1.2 kW, 2450 MHz resonant continuous microwave processing system was designed and optimized for oil extraction from green algae (Scenedesmus obliquus). Algae-water suspension (1:1 w/w) was heated to 80 and 95°C, and subjected to extraction for up to 30 min. Maximum oil yield was achieved at 95°C and 30 min. The microwave system extracted 76-77% of total recoverable oil at 20-30 min and 95°C, compared to only 43-47% for water bath control. Extraction time and temperature had significant influence (p<0.0001) on extraction yield. Oil analysis indicated that microwaves extracted oil containing higher percentages of unsaturated and essential fatty acids (indicating higher quality). This study validates for the first time the efficiency of a continuous microwave system for extraction of lipids from algae. Higher oil yields, faster extraction rates and superior oil quality demonstrate this system's feasibility for oil extraction from a variety of feedstock.