Influence of Spectral Range and Carbon and Nitrogen Sources on Oxygen Evolution and Emerson Enhancement in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii was grown in medium with different carbon (acetate, CO₂, or both), and nitrogen (ammonium chloride, peptone, urea) sources and under light of different spectral composition. The light-dark cycles were found more suitable for mixotrophic growth than continuous irradiation. Both blue (BR) and red (RR) radiations decreased photosynthetic capacity of mixotrophic cells compared to “white light” (WL). Effect of RR was associated with photon distribution favouring photosystem 1 (PS1) suggesting increased cyclic phosphorylation. Mixotrophic growth in 10 mM NH₄Cl increased photosynthetic oxygen evolution compared to standard concentration of 5 mM NH₄Cl used for growing C. reinhardtii. Autotrophic growth stimulated the photosynthetic capacity compared to mixotrophic one. However, higher photosynthetic capacity was achieved for mixotrophic cells by growing them at high NH₄ ⁺/K⁺ ratio and high phosphate concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

In Vitro Evolution and Preliminary Characterization of a Cadmium-Resistant Population of Chlamydomonas reinhardtii

A cadmium-tolerant population of Chlamydomonas reinhardtii was derived from a Cd-sensitive cell wall-deficient strain by long-term selection in liquid culture. A comparison of Cd-sensitive and Cd-tolerant cells revealed that Cd tolerance was due to genetically determined alterations of metabolism rather than to increased efficiency of a detoxification system.     

A mutant of Chlamydomonas reinhardtii altered in the transport of ammonium and methylammonium

Summary A methylammonium-resistant mutant, named hereafter strain 2170 (ma-1), was isolated for the first time from a eukaryotic phototrophic organism. Mutant 2170 from Chlamydomonas reinhardtii carries a single mendelian mutation which results in a decreased rate of uptake of both ammonium and methylammonium without being affected either in uptake of nitrate or nitrite or any of the tested enzyme activities related to ammonium assimilation. Mutant cells could not use methylammonium as nitrogen source nor excrete ammonium into the medium but they had derepressed nitrate and nitrite reductases when growing in the presence of ammonium. Mutant 2170 also exhibited a diminished methylammonium transport rate in comparison with the wild-type cells. We conclude that mutant 2170 is affected in a transport system responsible for the entrance of both ammonium and methylammonium into the cells.Abbreviations CHES 2-(N-Cyclohexylamino)ethanesulphonic acid - MOPS 3(N-morpholine)propanesulphonic acid     

The effect of oxygen concentration on photosynthetic biomass production by algae

The effect of decreased oxygen concentration on photosynthetic biomass production was determined for Euglena gracilis Klebs strain z and Chlamydomonas reinhardtii Dangeard. At a constant carbon dioxide concentration of 0.03% (v/v), decreasing the oxygen concentration from 21% to 2% (v/v) gave a two-fold increase in dry-weight yield for E. gracilis; a result consistent with the operation of a functional glycollate pathway in this alga. A similar effect of oxygen concentration on dry-weight yield was not observed with C. reinhardtii.     

Altered expression of the chloroplast ATP synthase through site-directed mutagenesis in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The chloroplast ATP synthase gates the flow of protons out of the thylakoid lumen. In Chlamydomonas reinhardtii deletion of any of the genes for the ATP synthase subunits, or misfolding of the peptides results in photosynthetic membranes devoid of the enzyme (Lemaire and Wollman, J Biol Chem 264:675–685, 1989). This work examines the physiologic response of an algal strain in which the epsilon subunit of the chloroplast ATP synthase has been truncated. Removal of 10 amino acids from the C-terminus of the peptide results in a sharp decrease in the content of the enzyme, but does not result in its exclusion from the thylakoid membranes. The ATP synthase of this mutant strain has a higher rate of ATP hydrolysis than the wild-type enzyme. This strain of C. reinhardtii exhibits reduced growth in the light, dependence on acetate, and a low threshold for the onset of photoinhibition. The role of the ATP synthase in regulating the proton concentration of the lumen is discussed. This work was supported in part by a grant from the National Science Foundation (MCB0110232).     

Distribution and functional role of carbonic anhydrase Cah3 associated with thylakoid membranes in the chloroplast and pyrenoid of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Localization of lumenal carbonic anhydrase Cah3 in thylakoid membranes of Chlamydomonas reinhardtii was studied using wild-type algae and photosynthetic mutants with different composition of chlorophyll-protein complexes in the photosystems. In addition, the photosynthetic characteristics of wild-type C. reinhardtii and cia3 mutants lacking the activity of carbonic anhydrase Cah3 were examined. Western blot analysis revealed the lack of cross reaction with antibodies to Cah3 in the mutant lacking the photosystem II (PSII) reaction center, in contrast to the mutant deficient in light-harvesting complex of PSII. These data show that the lumenal Cah3 is associated with polypeptides on the donor side of PSII reaction center. Using immunoelectron microscopy and antibodies to Cah3 from C. reinhardtii, we showed for the first time that the major part of thylakoid Cah3 is localized in the pyrenoid where the bulk of Rubisco is located. The rate of photosynthetic oxygen evolution and PSII photochemical efficiency were lower in C. reinhardtii cia3 mutant than in the wild type, especially in the cells grown at limiting CO₂ concentrations. These observations show that Cah3 takes part in CO₂-concentrating mechanism of the chloroplast. The results support our hypothesis [1, 2] that the carboxylation reaction in microalgae proceeds in the pyrenoid, a specific Rubisco-containing part of the chloroplast, which acquires CO₂ from the lumen of intrapyrenoid thylakoids. We discuss significance of the pyrenoid as an autonomous metabolic microcompartment, in which Cah3 plays a key role in the production and concentration of CO₂ for Rubisco. These functions may promote the photosynthetic efficiency owing to the effective CO₂ supply for the Calvin cycle.     

Functional characterization of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> with alterations in the <Emphasis Type="Italic">atpE</Emphasis> gene

The FUD17 strain of Chlamydomonas reinhardtii is a photosynthesis-deficient, acetate-requiring mutant with a defect in the chloroplast atpE gene, which codes for the ε subunit of the chloroplast ATP synthase. In this work, the FUD17 mutant was examined in relation to other known ATP synthase mutants as an initial step toward using this strain to generate altered versions of the atpE gene for site-directed mutagenesis of the ε subunit. The FUD17 strain grows well and is normally pigmented in the dark (heterotrophic conditions), but cannot grow autotrophically in the light, even when media are supplemented with acetate. Under heterotrophic conditions, it shows no accumulation of the ε subunit, and much lower levels of the α and β subunits of the chloroplast ATP synthase. FUD17 shows no light-dependent oxygen evolution and shows a strong, light-dependent alteration in its chlorophyll fluorescence. These results show that FUD17 possesses similar characteristics to other ATP synthase mutants and fails to express an assembled ATP synthase complex on its thylakoid membrane. A preliminary attempt at site-directed mutagenesis is described which produced a slightly truncated form of the ε subunit, which is expressed normally in the cell. This revised version was published online in June 2006 with corrections to the Cover Date.     

A Reduced Activity of Catalase as a Basis for Light Dependent Methionine Sensitivity of a Chlamydomonas reinhardtii Mutant

Pre-illumination of methionine-supplemented medium enhancedthe inactivation of the light dependent methionine sensitivemutant cells of Chlamydomonas reinhardtii and, to a lesser extent,the wild-type cells, confirming that the photodamage is dueto production of toxic produces) in the medium. Exogenouslyadded catalase protected the mutant cells from growth inhibition.Starch gel electrophoresis showed lower catalase activity inthe mutant cells than the wild type. Catalase activity whichwas followed by measuring O₂ evolution after the addition ofH₂O₂ to the cell suspensions was consistently lower in the mutantthan the wild type. The results indicate that light sensitivityin the presence of methionine expressed by this mutant is dueto reduced activity of catalase. (Received August 31, 1989; Accepted October 9, 1989)     

Photosystem II in a mutant of Chlamydomonas reinhardtii lacking the 23 kDa psbP protein shows increased sensitivity to photoinhibition in the absence of chloride

The psbP gene product, the so called 23 kDa extrinsic protein, is involved in water oxidation carried out by Photosystem II. However, the protein is not absolutely required for water oxidation. Here we have studied Photosystem II mediated electron transfer in a mutant of Chlamydomonas reinhardtii, the FUD 39 mutant, that lacks the psbP protein. When grown in dim light the Photosystem II content in thylakoid membranes of FUD 39 is approximately similar to that in the wild-type. The oxygen evolution is dependent on the presence of chloride as a cofactor, which activates the water oxidation with a dissociation constant of about 4 mM. In the mutant, the oxygen evolution is very sensitive to photoinhibition when assayed at low chloride concentrations while chloride protects against photoinhibition with a dissociation constant of about 5 mM. The photoinhibition is irreversible as oxygen evolution cannot be restored by the addition of chloride to inhibited samples. In addition the inhibition seems to be targeted primarily to the Mn-cluster in Photosystem II as the electron transfer through the remaining part of Photosystem II is photoinhibited with slower kinetics. Thus, this mutant provides an experimental system in which effects of photoinhibition induced by lesions at the donor side of Photosystem II can be studied in vivo.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - DPC 2,2-diphenylcarbonic dihydrazide - HEPES 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - P₆₈₀ the primary electron donor to PS II - PpBQ phenyl-p-benzoquinone - PS II Photosystem II - Q_A the first quinone acceptor of PS II - Q_B the second quinone acceptor of PS II - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)aminomethane - Tyr_D accessory electron donor on the D₂-protein - Tyr_Z tyrosine residue, acting as electron carrier between P₆₈₀ and the water oxidizing system     

Evidence of singlet oxygen evolution by whole living cells of Chlamydomonas reinhardtii

The oxygen evolved by Chlamydomonas reinhardtii in the light is measured simultaneously with a Clark electrode and with the nitrosodimethylaniline-imidazole colorimetric method which is specific for singlet oxygen. Experiments with wild-type and FuD7 mutant cells (unable to synthesize the D1 protein of Photosystem II), with dichlorophenyldimethylurea (which blocks electron transfer from Photosystem II to Photosystem I) and with dibromothymoquinone (which diverts electrons from their normal path between the two photosystems), as well as with hydroxylamine (an inactivator of the water-splitting part of Photosystem II and a competitor of water for electron donation to it), all point to the dependence of detected singlet oxygen on photolysis of water by Photosystem II.Abbreviations DBMIB Dibromothymoquinone - DCMU Dichlorophenyldimethylurea - PS I and PS II Photosystems I and II - RNO para-nitrosodimethylaniline Contribution of the Centre interdisciplinaire de Biochimie de Oxygène.     

Screening of oleaginous algal strains from Chlamydomonas reinhardtii mutant libraries via density gradient centrifugation

Microalgae have a high potential to be utilized as feedstock for biofuels because they have high growth rates and do not compromise food production. Commercialized algae-based biofuel production relies on the development of strains with high lipid content. Based on the relatively low density of lipids compared to other cellular components, density gradient centrifugation was used to isolate high lipid content algal strains from Chlamydomonas reinhardtii mutant libraries. The correlation between cell density and lipid content was confirmed by analysis of Nile red fluorescence intensity, total lipids, and total fatty acid methyl ester content. A strain isolated by this screening method had 50% higher lipid content and 7% lower cell density than the parent wild-type strain. Consequently, we demonstrated that screening of algal strains with low cell density via continuous density gradient centrifugation allows simple, rapid, and inexpensive screening for high lipid content strains.     

Growth and Heavy Metal Binding Properties of Transgenic Chlamydomonas Expressing a Foreign Metallothionein Gene

We have compared the growth rates and cadmium binding capacity of wild-type and transgenic Chlamydomonas reinhardtii cells expressing a foreign class-II metallothionein. We observed that cells expressing metallothionein grew to significantly higher cell densities than wild-type cells in the presence of a toxic cadmium concentration (40 μM). When grown at a low (5 μM) cadmium concentration, cells expressing metallothionein bound twofold more cadmium (0.43 μg Cd)mg Ch1) than wild-type. At cadmium concentrations (40 μM), which induce phytochelatin synthesis in wild-type cells the cadmium binding capacity of both wild-type (79.6 μg Cd)mg Ch1) and transformed cells (86.4 μg Cd)mg Ch1) was similar; however, the transformed cells grew to higher densities than the wild type. These results suggest that under conditions that apparently induce phytochelatin expression, the presence of metallothionein in the cytoplasm reduces heavy metal toxicity. Furthermore, because cells expressing metallothionein grow to higher densities than wild-type cells at a toxic cadmium concentration (40 μM), the transgenic cells sequester more total cadmium (9% of total Cd) from the medium than the wild type (5.5% of total Cd). These results indicate that the trace-metal binding properties of Chlamydomonas can be enhanced through the expression of trace-metal-specific binding proteins.     

CHLOROPLAST STRUCTURE AND FUNCTION IN ac-20, A MUTANT STRAIN OF CHLAMYDOMONAS REINHARDI : III. Chloroplast Ribosomes and Membrane Organization

The fine structure of the ac-20 strain of Chlamydomonas reinhardi is described. Cells grown mixotrophically in the presence of acetate have a highly disordered chloroplast membrane organization and usually lack pyrenoids. Chloroplast ribosome levels are only 5–10% of wild-type levels. Cells grown phototrophically without acetate possess more chloroplast ribosomes and have more normal membrane and pyrenoid organization. Chloroplast ribosome levels rise rapidly when cells are transferred from acetate to minimal medium, whereas membrane reorganization occurs only after a lag. These results, combined with earlier studies of the photosynthetic properties of the mutant strain, suggest that proper membrane organization, Photosystem II activity, and ribulose-1,5-diphosphate carboxylase formation are dependent on the presence of chloroplast ribosomes. Other chloroplast components tested are unaffected by a 10-fold reduction in levels of chloroplast ribosomes.     

Triacylglyceride Production and Autophagous Responses in Chlamydomonas reinhardtii Depend on Resource Allocation and Carbon Source

To improve the economic viability of microalgal biodiesel, it will be essential to optimize the productivity of fuel molecules such as triacylglyceride (TAG) within the microalgal cell. To understand some of the triggers required for the metabolic switch to TAG production, we studied the effect of the carbon supply (acetate or CO₂) in Chlamydomonas reinhardtii (wild type and the starchless sta6 mutant) grown under low N availability. As expected, initial rates of TAG production were much higher when acetate was present than under strictly photosynthetic conditions, particularly for the sta6 mutant, which cannot allocate resources to starch. However, in both strains, TAG production plateaued after a few days in mixotrophic cultures, whereas under autotrophic conditions, TAG levels continued to rise. Moreover, the reduced growth of the sta6 mutant meant that the greatest productivity (measured as mg TAG liter⁻¹ day⁻¹) was found in the wild type growing autotrophically. Wild-type cells responded to low N by autophagy, as shown by degradation of polar (membrane) lipids and loss of photosynthetic pigments, and this was less in cells supplied with acetate. In contrast, little or no autophagy was observed in sta6 mutant cells, regardless of the carbon supply. Instead, very high levels of free fatty acids were observed in the sta6 mutant, suggesting considerable alteration in metabolism. These measurements show the importance of carbon supply and strain selection for lipid productivity. Our findings will be of use for industrial cultivation, where it will be preferable to use fast-growing wild-type strains supplied with gaseous CO₂ under autotrophic conditions rather than require an exogenous supply of organic carbon.     

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.     

Photoresponses of wild-type and mutant dikaryons ofChlamydomonas

Photoaccumulation and random motility of wild-type and mutant gametes and dikaryons ofChlamydomonas reinhardtii were evaluated with quantitative assays and compared with those of vegetative cells. Gametes exhibited behavior similar to that of vegetative cells. Dikaryons constructed from (+) and (−) wild-type gametes exhibited strong photoaccumulation in the presence of a stimulus and normal random swimming in red light, which shows that the activity of flagella and other components from two cells can be integrated and coordinated to permit appropriate behavior. Dikaryons from crosses of the wild type with mutants exhibited intermediate photoaccumulation. suggesting that neither phenotype is dominant. In contrast, crosses between an abnormally swimming mutant and normally motile strains showed that wild-type swimming was dominant. Partial complementation of mutant photoresponse phenotypes occurred in some crosses, but recovery of fully normal behavior was not observed.     

The carboxy-terminal extension of the D1-precursor protein is dispensable for a functional photosystem II complex in Chlamydomonas reinhardtii

The D1-precursor protein of the photosystem II reaction centre contains a carboxy-terminal extension whose proteolytic removal is necessary for oxygen-evolving activity. To address the question of the role of the carboxy-terminal extension in the green alga Chlamydomonas reinhardtii, we truncated D1 by converting codon Ser345 of the psbA gene into a stop codon. Particle gun transformation of an in vitro modified psbA gene fragment also carrying mutations conferring herbicide resistance yielded a homoplasmic transformant containing the stop codon. Since oxygen evolution capacity is not affected in this mutant as compared with herbicide-resistant control cells, the carboxy-terminal extension is dispensable for a functional photosystem II complex under normal growth conditions.     

Chloroplast sulfate transport in green algae – genes,proteins and effects

This review summarizes evidence at the molecular genetic, protein and regulatory levels concerning the existence and function of a putative ABC-type chloroplast envelope-localized sulfate transporter in the model unicellular green alga Chlamydomonas reinhardtii. From the four nuclear genes encoding this sulfate permease holocomplex, two are coding for chloroplast envelope-targeted transmembrane proteins (SulP and SulP2), a chloroplast stroma-targeted ATP-binding protein (Sabc) and a substrate (sulfate)-binding protein (Sbp) that is localized on the cytosolic side of the chloroplast envelope. The sulfate permease holocomplex is postulated to consist of a SulP–SulP2 chloroplast envelope transmembrane heterodimer, flanked by the Sabc and the Sbp proteins on the stroma side and the cytosolic side of the inner envelope, respectively. The mature SulP and SulP2 proteins contain seven transmembrane domains and one or two large hydrophilic loops, which are oriented toward the cytosol. The corresponding prokaryotic-origin genes (SulP and SulP2) probably migrated from the chloroplast to the nuclear genome during the evolution of Chlamydomonas reinhardtii. These genes, or any of its homologues, have not been retained in vascular plants, e.g. Arabidopsis thaliana, although they are encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). The function of the SulP protein was probed in antisense transformants of C. reinhardtii having lower expression levels of the SulP gene. Results showed that cellular sulfate uptake capacity was lowered as a consequence of attenuated SulP gene expression in the cell, directly affecting rates of de novo protein biosynthesis in the chloroplast. The antisense transformants exhibited phenotypes of sulfate-deprived cells, displaying slow rates of light-saturated oxygen evolution, low levels of Rubisco in the chloroplast and low steady-state levels of the Photosystem II D1 reaction center protein. The role of the chloroplast sulfate transport in the uptake and assimilation of sulfate in Chlamydomonas reinhardtii is discussed along with its impact on the repair of Photosystem II from a frequently occurring photo-oxidative damage and H₂-evolution related metabolism in this green alga.     

CDKA and CDKB kinases from Chlamydomonas reinhardtii are able to complement cdc28 temperature-sensitive mutants of Saccharomyces cerevisiae

Summary. Cyclin-dependent kinases (CDK) play a key role in coordinating cell division in all eukaryotes. We investigated the capability of cyclin-dependent kinases CDKA and CDKB from the green alga Chlamydomonas reinhardtii to complement a Saccharomyces cerevisiae cdc28 temperature-sensitive mutant. The full-length coding regions of algal CDKA and CDKB cDNA were amplified by RT-PCR and cloned into the yeast expression vector pYES-DEST52, yielding pYD52-CDKA and pYD52-CDKB. The S. cerevisiae cdc28-1N strain transformed with these constructs exhibited growth at 36 °C in inducing (galactose) medium, but not in repressing (glucose) medium. Microscopic observation showed that the complemented cells had the irregular cylindrical shape typical for G₂ phase-arrested cells when grown on glucose at 36 °C, but appeared as normal budded cells when grown on galactose at 36 °C. Sequence analysis and complementation tests proved that both CDKA and CDKB are functional CDC28/cdc2 homologs in C. reinhardtii. The complementation of the mitotic phenotype of the S. cerevisiae cdc28-1N mutant suggests a mitotic role for both of the kinases. Correspondence: K. Bišová, Laboratory of Cell Cycles of Algae, Institute of Microbiology, Academy of Sciences of the Czech Republic, 379 81 Třeboň, Czech Republic.     

Effect of dissolved inorganic carbon on oxygen evolution and uptake by Chlamydomonas reinhardtii suspensions adapted to ambient and CO2-enriched air

Mass spectrometric measurements of ¹⁶O₂ and ¹⁸O₂ isotopes were used to compare the rates of gross O₂ evolution (E₀), O₂ uptake (U₀) and net O₂ evolution (NET) in relation to different concentrations of dissolved inorganic carbon (DIC) by Chlamydomonas reinhardtii cells grown in air (air-grown), in air enriched with 5% CO₂ (CO₂-grown) and by cells grown in 5% CO₂ and then adapted to air for 6h (air-adapted).At a photon fluence rate (PFR) saturating for photosynthesis (700 mol photons m^-2 s^-1), pH=7.0 and 28°C, U₀ equalled E₀ at the DIC compensation point which was 10M DIC for CO₂-grown and zero for air-grown cells. Both E₀ and U₀ were strongly dependent on DIC and reached DIC saturation at 480 M and 70 M for CO₂-grown and air-grown algae respectively. U₀ increased from DIC compensation to DIC saturation. The U₀ values were about 40 (CO₂-grown), 165 (air-adapted) and 60 mol O₂ mg Chl^-1 h^-1 (air-grown). Above DIC compensation the U₀/E₀ ratios of air-adapted and air-grown algae were always higher than those of CO₂-grown cells. These differences in O₂ exchange between CO₂- and air-grown algae seem to be inducable since air-adapted algae respond similarly to air-grown cells.For all algae, the rates of dark respiratory O₂ uptake measured 5 min after darkening were considerably lower than the rates of O₂ uptake just before darkening. The contribution of dark respiration, photorespiration and the Mehler reaction to U₀ is discussed and the energy requirement of the inducable CO₂/HCO₃ ^- concentrating mechanism present in air-adapted and air-grown C. reinhardtii cells is considered.Abbreviations DIC dissolved inorganic carbon - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - E₀ rate of photosynthetic gross O₂ evolution - PCO photosynthetic carbon oxidation - PFR photon fluence rate - PS I photosystem I - PS II photosystem II - U₀ rate of O₂ uptake in the light - MS mass spectrometer