Strains Of Chlamydomonas Research Articles

Isolation and selection of microalgae capable of stimulating rice plant development and seed production

The growing global demand for food, in addition to the environmental impacts of agricultural intensification and climate change, reinforce the need for sustainable alternatives to maintain and increase agricultural productivity. Microalgae are widely available renewable resources for application in agricultural environments. There is a vast biodiversity of microalgae species, and their versatile metabolism produces a great diversity of compounds that favor the capture and supply of nutrients, improve soil characteristics, and stimulate plant growth. In this study, we evaluated the production of indole compounds, exopolysaccharides, siderophores, and the development capacity of microalgae strains of the genera Chlamydomonas sp., Chlorella sp. and Desmodesmus sp. isolated from different Brazilian States. Microalgae that produced a greater range of compounds and developed more quickly were selected for the production of aqueous extracts from their biomass in order to evaluate their potential as a biostimulant for rice (Oryza sativa L.) plants. Seed inoculation and foliar application of extracts stimulated plant development. Shoot length in seedlings 7 days after germination was increased after the application of Syn 16 aqueous extract at concentration of 0.5 g/L. Shoots of 30-day-old plants increased in length with the application of Syn 16 and Syn 90 strains, regardless of the tested concentration, while root length was increased by Syn 16 (at both concentrations - 0.1 and 0.5 g/L) and Syn 90 (0.1 g/L). Regarding shoot dry weight, increases of up to 36 % were detected with the Syn 16 strain at concentration of 0.5 g/L. With the Syn 90 strain at a concentration of 0.5 g/L, the increase was 27 %. Furthermore, root dry weight was 43 % and 35 % higher than control plants after the application of Syn 16 and Syn 90, respectively (both at 0.5 g/L concentration). At the maturity stage, application of Syn 46 strain (0.5 g/L concentration) increased 11 % the number of full seeds per panicle when compared to control condition. Our results demonstrate the ability of microalgae extracts in stimulating the development and seed production of rice plants.

Comparative Phenotyping of Two Commonly Used Chlamydomonas reinhardtii Background Strains: CC-1690 (21gr) and CC-5325 (The CLiP Mutant Library Background).

The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to investigate many essential cellular processes in photosynthetic eukaryotes. Two commonly used background strains of Chlamydomonas are CC-1690 and CC-5325. CC-1690, also called 21gr, has been used for the Chlamydomonas genome project and several transcriptome analyses. CC-5325 is the background strain for the Chlamydomonas Library Project (CLiP). Photosynthetic performance in CC-5325 has not been evaluated in comparison with CC-1690. Additionally, CC-5325 is often considered to be cell-wall deficient, although detailed analysis is missing. The circadian rhythms in CC-5325 are also unclear. To fill these knowledge gaps and facilitate the use of the CLiP mutant library for various screens, we performed phenotypic comparisons between CC-1690 and CC-5325. Our results showed that CC-5325 grew faster heterotrophically in dark and equally well in mixotrophic liquid medium as compared to CC-1690. CC-5325 had lower photosynthetic efficiency and was more heat-sensitive than CC-1690. Furthermore, CC-5325 had an intact cell wall which had comparable integrity to that in CC-1690 but appeared to have reduced thickness. Additionally, CC-5325 could perform phototaxis, but could not maintain a sustained circadian rhythm of phototaxis as CC1690 did. Finally, in comparison to CC-1690, CC-5325 had longer cilia in the medium with acetate but slower swimming speed in the medium without nitrogen and acetate. Our results will be useful for researchers in the Chlamydomonas community to choose suitable background strains for mutant analysis and employ the CLiP mutant library for genome-wide mutant screens under appropriate conditions, especially in the areas of photosynthesis, thermotolerance, cell wall, and circadian rhythms.

Chlamydomonas strains respond differently to photoproduction of hydrogen and by-products and nutrient uptake in sulfur-deprived cultures

Hydrogen is a renewable and clean energy carrier that can be generated from microalgae. This study aimed to investigate the hydrogen and by-product production and nutrient uptake by five different strains of Chlamydomonas, including a wild-type strain. Selected strains were cultivated in two phases under continuous illumination: the first step was to obtain biomass; in the second step, the biomass was transferred to closed anaerobic photobioreactors for hydrogen generation under sulfur deprivation. There was an effect of different strains of Chlamydomonas on hydrogen and fermentative metabolite production and nutrient uptake. Wild-type strain had the best hydrogen productivity (9.23 µmol H2 mg Chl a-1 h-1), followed by C. reinhardtii CC425 strain (3.45 µmol H2 mg Chl a-1 h-1) and this result was on average 16 and 6 times higher, respectively, than the other strains. Strains that produced less hydrogen had better ethanol production (average of 280.6 mg L-1). The CC425 strain presented the best nutrient uptake efficiency, mainly of phosphate (67%). The results confirmed significant differences in anoxic metabolism from sulfur-deprivation-induced and metabolic flexibility by Chlamydomonas genus. Although all strains generated products of interest, the species, different strains, and cultivation conditions must be considered according to the purpose of the study.

Comparative secretome analysis between salinity-tolerant and control Chlamydomonas reinhardtii strains.

Secretome analysis of a salt-tolerant and control Chlamydomonas reinhardtii revealed 514 differentially expressed proteins. Membrane transport and trafficking, signal transduction and channel proteins were up-regulated in the ST secretome. Salinity is a major abiotic stress that limits crop production worldwide. Multiple adverse effects have been reported in many living organisms exposed to high-saline concentrations. Chlamydomonas reinhardtii is known for secreting proteins in response to many environmental stresses. A salinity-tolerant (ST) strain of Chlamydomonas has been developed, whose cells were able to grow at 300mM NaCl. The current study analyzed the secretomes of ST grown in TAP medium supplemented with 300mM NaCl and the laboratory strain CC-503 grown in TAP medium without NaCl supplement. In total, 514 secreted proteins were identified of which 203 were up-regulated and 110 were down-regulated. Bioinformatic analysis predicted 168 proteins to be secreted or in the conventional secretory pathway. Out of these, 70 were up-regulated, while 51 proteins were down-regulated. Proteins involved in membrane transport and trafficking, signal transduction and channel proteins were altered in their expression in the ST secretome, suggesting the response of saline stress acts toward not only the intracellular pool of proteins but also the extracellular proteins. This also suggested that the secreted proteins might have roles in the extracellular space. Signal peptide (SP) prediction revealed that almost 40% of the predicted secreted proteins contained a signal peptide; however, a high proportion of proteins lacked an SP, suggesting that these proteins might be secreted through an unconventional protein secretion pathway.

Resistive force theory and wave dynamics in swimming flagellar apparatus isolated from C. reinhardtii.

Cilia-driven motility and fluid transport are ubiquitous in nature and essential for many biological processes, including swimming of eukaryotic unicellular organisms, mucus transport in airway apparatus or fluid flow in the brain. The-biflagellated micro-swimmer Chlamydomonas reinhardtii is a model organism to study the dynamics of flagellar synchronization. Hydrodynamic interactions, intracellular mechanical coupling or cell body rocking is believed to play a crucial role in the synchronization of flagellar beating in green algae. Here, we use freely swimming intact flagellar apparatus isolated from a wall-less strain of Chlamydomonas to investigate wave dynamics. Our analysis on phase coordinates shows that when the frequency difference between the flagella is high (10–41% of the mean), neither mechanical coupling via basal body nor hydrodynamics interactions are strong enough to synchronize two flagella, indicating that the beating frequency is perhaps controlled internally by the cell. We also examined the validity of resistive force theory for a flagellar apparatus swimming freely in the vicinity of a substrate and found quantitative agreement between the experimental data and simulations with a drag anisotropy of ratio 2. Finally, using a simplified wave form, we investigated the influence of phase and frequency differences, intrinsic curvature and wave amplitude on the swimming trajectory of flagellar apparatus. Our analysis shows that by controlling the phase or frequency differences between two flagella, steering can occur.

Tolerance Capacity of Chlamydomonas VHLR Mutants for the Toxicity of Mercury

The toxicity effect of metals on Chlamydomonas strains having a higher tolerance level for oxidative stress is important to investigate for better understanding their tolerance capacity. In this study, the toxicity of HgCl2 was determined on two very high light resistant (VHLR) mutant strains of Chlamydomonas, CC-3723 and CC-3724, and on the wild-type CC-125. All strains were exposed to 1–7 μM of HgCl2 during 24–72 h. The results showed that VHLR mutants were less affected by the effect of HgCl2 than CC-125, due to a better ability to detoxify the level of intracellular reactive oxygen species (ROS) generated by Hg. In particular, the effect of 3 μM of HgCl2 inhibited completely the growth rate of CC-125 at 24 h, which was correlated with a high accumulation of Hg and a strong level of ROS. From 48 to 72 h, the growth rate was recovered for all strains, which was stronger for VHLR mutants than the wild-type by 50% at 48 h, when compared to 24 h. In addition, the extracytoplasmic polyP level decreased significantly compared to the control for all strains treated to 3 μM of HgCl2 during 24–48 h. These results suggested that the polyP did participate to the tolerance for Hg through its cellular sequestration. Furthermore, the analysis of cellular morphology showed that VHLR mutants formed palmelloid colonies of 4–5 cells under 3 μM of HgCl2 at 48 h, reducing the surface contact with Hg. Therefore, VHLR mutants were more tolerant than the wild-type to the effect of low concentrations of HgCl2 (1 and 3 μM).

Isolation of a freshwater microalgae and its application for the treatment of wastewater and obtaining fatty acids from tilapia cultivation.

In the present study, isolated strains of the microalgae Chlamydomonas sp. (CH) and Chlorella vulgaris (CV) were used to treat aquaculture wastewater and to obtain fatty acids and from a fattening culture of tilapia. The microalgae were cultivated for 11days in tubular photobioreactors with an operating volume of 2L, constant aeration and illumination. High removal rates of NO3- and PO43- were achieved for both Chlamydomonas sp. (84.7% and 96%, respectively) and Chlorella vulgaris (94.6 and 97.9%, respectively). The maximum biomass productivity achieved by Chlamydomonas sp. was 0.06 and 0.10 gL-1d-1 for Chlorella vulgaris. Therefore, tilapia wastewater contained the necessary nutrient concentration for algal growth and development. Chlamydomonas sp. biomass lipid content was 69%, while that of Chlorella vulgaris was 40%. The lipid profile of both microalgae was abundant in palmitic acid (78% for Chlamydomonas sp. and 35% for Chlorella vulgaris). This fatty acid is suitable for biodiesel production. Tilapia wastewater is low-cost alternative culture medium as it contains the necessary nutrient concentration for microalgae development and growth.

Screening for hydrogen-producing strains of green microalgae in phosphorus or sulphur deprived medium under nitrogen limitation

Hydrogen gas, one of the best candidates for clean and renewable energy, can be produced by microalgae that can use solar energy to cause photolysis of water. This study screened H-2-producing indigenous green microalgae under sulphur (S-) deprivation, simultaneous nitrogen (N-) limitation and S-deprivation, or simultaneous N-limitation and phosphorus (P-) deprivation. Sequences of 18S rDNA and ITS in conjunction with morphological characteristics were used to identify the algae. We report that Chlorella lewinii, Micractinium sp., Coelastrella sp., and Monoraphidium sp. have the ability to produce H-2. The increase in H-2 photoproduction when N is limited seems to be a universal phenomenon in most tested strains of Chlorella, in all strains of Chlamydomonas, but in no strain of Coelastrella, Micractinium, or Scenedesmus. Chlorella sorokiniana KU204 produced H-2 (46 ml/l) under S-deprivation. This strain exhibited the highest H-2-producing ability (1.30 ml l(-1) h(-1)) and accumulated up to 90 ml/l under simultaneous N-limitation and S-deprivation. Interestingly, C. sorokiniana KU204 could also produce H-2 under simultaneous N-limitation and P-starvation (69 ml/l). The induction time to reach an anoxic state by most tested strains of Chlorella, but not Chlamydomonas, was shorter under simultaneous N-limitation and S-deprivation than under S-deprivation. In addition, those strains of Chlorella exhibited high H-2 photoproduction under simultaneous N-limitation and S-deprivation. A few Chlorella strains were unable to reach an anoxic state during the experiment. However, such regularity is not found in Chlamydomonas. The results indicate that the mechanism of H-2 photoproduction in Chlorella may differ from that in Chlamydomonas.

A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii

In search of alternative expression platforms heterologous protein production in microalgae has gained increasing importance in the last years. Particularly, the chloroplast of the green alga Chlamydomonas reinhardtii has been adopted to successfully express foreign proteins like vaccines and antibodies. However, when compared with other expression systems, the development of the algal chloroplast to a powerful production platform for recombinant proteins is still in its early stages. In an effort to further improve methods for a reliable and rapid generation of transplastomic Chlamydomonas strains we constructed the key plasmid pMM2 containing the psbA gene and a multiple cloning site for foreign gene insertion. The psbA gene allows a marker-free selection procedure using as a recipient the Fud7 strain of Chlamydomonas, which grows on media containing acetate as a carbon source, but is unable to grow photoautotrophically due to the lack of an intact psbA gene. Biolistic transformation of Fud7 with vectors containing this gene restores photoautotrophic growth and thus permits selection in the light on media without carbon sources and antibiotics. The multiple cloning site with a BsaI recognition sequence allows type IIs restriction enzyme-based modular cloning which rapidly generates new gene constructs without sequences, which could influence the expression and characteristics of the foreign protein. In order to demonstrate the feasibility of this approach, a codon optimized version of the gene for the bacterial protein MPT64 has been integrated into the plastome. Several strains with different promoter/UTR combinations show a stable expression of the HA tagged MPT64 protein in Chlamydomonas chloroplasts.

A high-throughput fatty acid profiling screen reveals novel variations in fatty acid biosynthesis in Chlamydomonas reinhardtii and related algae.

Analysis of fatty acid methyl esters (FAMEs) by gas chromatography (GC) is a common technique for the quantitative and qualitative analysis of acyl lipids. Methods for FAME preparation are typically time-consuming and labor-intensive and require multiple transfers of reagents and products between reaction tubes and autosampler vials. In order to increase throughput and lower the time and materials costs required for FAME preparation prior to GC analysis, we have developed a method in which 10-to-20-mg samples of microbial biomass are transferred to standard GC autosampler vials, transesterified using an emulsion of methanolic trimethylsulfonium hydroxide and hexane, and analyzed directly by GC without further sample handling. This method gives results that are essentially identical to those obtained by the more labor- and material-intensive FAME preparation methods, such as transmethylation with methanolic HCl. We applied this method to the screening of laboratory and environmental isolates of the green alga Chlamydomonas for variations in fatty acid composition. This screening method facilitated two novel discoveries. First, we identified a common laboratory strain of C. reinhardtii, CC-620, completely lacking all ω-3 fatty acids normally found in this organism and showed that this strain contains an inactivating mutation in the CrFAD7 gene, encoding the sole ω-3 desaturase activity in this organism. Second, we showed that some species of Chlamydomonas make Δ6-unsaturated polyunsaturated fatty acids (PUFA) rather than the Δ5 species normally made by the previously characterized laboratory strains of Chlamydomonas, suggesting that there is species-specific variation in the regiospecificity and substrate selectivity of front-end desaturases in this algal genus.

Detection and characterization of phosphatidylcholine in various strains of the genus Chlamydomonas (Volvocales, Chlorophyceae).

The laboratory strains of Chlamydomonas reinhardtii have been reported to contain no phosphatidylcholine (PC), which is considered to be replaced by another zwitterionic lipid, diacylglyceryl-N,N,N-trimethylhomoserine (DGTS). According to the recently published classification, the strains belonged to the subgroup Reinhardtinia. Screening for PC in 13 selected strains of Chlamydomonas in the NIES Algal Collection, which are different in habitats and belong to different phylogenetic subgroups in the genus, revealed the presence of PC in four strains: a strain in the subgroup Polytominia, and three strains in Reinhardtinia. PC was not detected in three other strains of Reinhardtinia analyzed. The presence/absence of PC was not related to the phylogenetic relationship based on 18S rRNA. DGTS was detected in all the strains analyzed. The rare isomer of linolenic acid, 18:3(5,9,12), which has been found in the DGTS of C. reinhardtii, was found in the PC of the two strains and in the DGTS of the five strains. The occurrence of this fatty acid seems limited to a branch of Reinhardtinia. Acquisition and loss of PC in various strains of Chlamydomonas are discussed from the viewpoint of evolution of PC biosynthetic pathway.

Genetic correlation in relation to differences in dosage of a stressor

We exposed the strains of Chlamydomonas isolated from an outbred laboratory population to a range of concentrations of salt (NaCl) up to an extirpative level that the base population could not tolerate. The genetic variance of yield increased with stress over the first half of this range before collapsing to nearly zero. The genetic correlation decreased with environmental distance, whether measured as a difference in dosage or as an environmental variance. This result is consistent with previous studies and provides a basis for interpreting adaptation to a deteriorating environment and the process of evolutionary rescue.

Critical Role of 7,8-Didemethyl-8-hydroxy-5-deazariboflavin for Photoreactivation in Chlamydomonas reinhardtii

DNA photolyases use two noncovalently bound chromophores to catalyze photoreactivation, the blue light-dependent repair of DNA that has been damaged by ultraviolet light. FAD is the catalytic chromophore for all photolyases and is essential for photoreactivation. The identity of the second chromophore is often 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO). Under standard light conditions, the second chromophore is considered nonessential for photoreactivation because DNA photolyase bound to only FAD is sufficient to catalyze the repair of UV-damaged DNA. phr1 is a photoreactivation-deficient strain of Chlamydomonas. In this work, the PHR1 gene of Chlamydomonas was cloned through molecular mapping and shown to encode a protein similar to known FO synthases. Additional results revealed that the phr1 strain was deficient in an FO-like molecule and that this deficiency, as well as the phr1 photoreactivation deficiency, could be rescued by transformation with DNA constructs containing the PHR1 gene. Furthermore, expression of a PHR1 cDNA in Escherichia coli produced a protein that generated a molecule with characteristics similar to FO. Together, these results indicate that the Chlamydomonas PHR1 gene encodes an FO synthase and that optimal photoreactivation in Chlamydomonas requires FO, a molecule known to serve as a second chromophore for DNA photolyases.

Chlamydomonas as a “new” organism for biodiesel production

The production of biodiesel from a naturally isolated strain of Chlamydomonas was investigated. The microalgal strain was isolated from the rice paddy-field soil samples during a screening program. The identification was done using physiological and molecular approaches. After reaching the stationary phase of growth, the total content of the lipids was extracted. The extracted fatty acids were primarily esterified and then identified through TLC and GC/MS analysis. Several types of fatty acid methyl esters (FAMEs) were identified in the isolated microalga and the presence of at least nine FAMEs in Chlamydomonas sp. MCCS 026 was shown. The total fatty acid content of the isolated strain was 25%. The composition of fatty acids in the studied species of microalga was mainly docosanoic acid methyl ester, tetradecanoic acid methyl ester, hexadecanoic acid methyl ester and nonanoic acid methyl ester.

Replacement of α-Tocopherol by β-Tocopherol Enhances Resistance to Photooxidative Stress in a Xanthophyll-Deficient Strain of Chlamydomonas reinhardtii

Tocopherols (vitamin E) comprise a class of lipid-soluble antioxidants synthesized only in plants, algae, and some cyanobacteria. The majority of tocopherols in photosynthetic cells is in the alpha form, which has the highest vitamin E activity in humans, whereas the beta, gamma, and delta forms normally account for a small percentage of total tocopherols. The antioxidant activities of these forms of tocopherol differ depending on the experimental system, and their relative activities in vivo are unclear. In a screen for suppressors of the xanthophyll-deficient npq1 lor1 double mutant of Chlamydomonas reinhardtii, we isolated a vte3 mutant lacking alpha-tocopherol but instead accumulating beta-tocopherol. The vte3 mutant contains a mutation in the homolog of a 2-methyl-6-phytyl-1,4-benzoquinone methyltransferase gene found in plants. The vte3 npq1 lor1 triple mutant with beta-tocopherol survived better under photooxidative stress than did the npq1 lor1 mutant, but the vte3 mutant on its own did not have an obvious phenotype. Following transfer from low light to high light, the triple mutant showed a higher efficiency of photosystem II, a higher level of cell viability, and a lower level of lipid peroxide, a marker for oxidative stress, than did the npq1 lor1 mutant. After high-light transfer, the level of the photosystem II reaction center protein, D1, was also higher in the vte3 npq1 lor1 mutant, but the rate of D1 photodamage was not significantly different from that of the npq1 lor1 mutant. Taken together, these results suggest that the replacement of alpha-tocopherol by beta-tocopherol in a xanthophyll-deficient strain of Chlamydomonas reinhardtii contributes to better survival under conditions of photooxidative stress.

Mechanism of REP27 protein action in the D1 protein turnover and photosystem II repair from photodamage.

The function of the REP27 protein (GenBank accession no. EF127650) in the photosystem II (PSII) repair process was elucidated. REP27 is a nucleus-encoded and chloroplast-targeted protein containing two tetratricopeptide repeat (TPR) motifs, two putative transmembrane domains, and an extended carboxyl (C)-terminal region. Cell fractionation and western-blot analysis localized the REP27 protein in the Chlamydomonas reinhardtii chloroplast thylakoids. A folding model for REP27 suggested chloroplast stroma localization for amino- and C-terminal regions as well as the two TPRs. A REP27 gene knockout strain of Chlamydomonas, termed the rep27 mutant, was employed for complementation studies. The rep27 mutant was aberrant in the PSII-repair process and had substantially lower than wild-type levels of D1 protein. Truncated REP27 cDNA constructs were made for complementation of rep27, whereby TPR1, TPR2, TPR1+TPR2, or the C-terminal domains were deleted. rep27-complemented strains minus the TPR motifs showed elevated levels of D1 in thylakoids, comparable to those in the wild type, but the PSII photochemical efficiency of these strains was not restored, suggesting that the functionality of the PSII reaction center could not be recovered in the absence of the TPR motifs. It is suggested that TPR motifs play a role in the functional activation of the newly integrated D1 protein in the PSII reaction center. rep27-complemented strains missing the C-terminal domain showed low levels of D1 protein in thylakoids as well as low PSII photochemical efficiency, comparable to those in the rep27 mutant. Therefore, the C-terminal domain is needed for a de novo biosynthesis and/or assembly of D1 in the photodamaged PSII template. We conclude that REP27 plays a dual role in the regulation of D1 protein turnover by facilitating cotranslational biosynthesis insertion (C-terminal domain) and activation (TPR motifs) of the nascent D1 during the PSII repair process.

Tolerance to cadmium in Chlamydomonas sp. (Chlorophyta) strains isolated from an extreme acidic environment, the Tinto River (SW, Spain)

The effects of selected concentrations of Cd on the growth and ultrastructure of three strains of Chlamydomonas sp. isolated from a highly acidic river, Río Tinto (SW Spain) were examined. The river is characterized by its extreme physico-chemical conditions in terms of low pH, mean 2.2 and high concentrations of heavy metals. Growth, Cd accumulation, chlorophyll a, influence of Fe in Cd toxicity and ultrastructural localization were determined. The strains were cultured in both, artificial chemically defined media as well as in natural water from the river. Since iron is the main component of the river water, the effect of different concentrations of this element in relation with Cd toxicity was also analysed. The three strains analysed showed comparable growth and ultrastructural changes. Cd concentration corresponding to 50% growth inhibition (EC 50) was 0.2 mM when cells were grown in artificial media. When cells were grown in natural water, no significant differences were found between the controls and the Cd supplemented media even at the highest concentration of 0.8 mM. At an inhibitory level of 0.1 mM of Cd, increasing the concentration of iron up to 90 or 180 mM resulted in a dramatic recovery in algal growth rates in artificial media, reaching normal growth curves. The accumulation of Cd depended on dose and time in the artificial media. The maximal accumulation of Cd was reached after 3 days for all Cd doses, and remained almost unchanged in the subsequent period of time. Chlorophyll a amount depended on dose but not on time in the artificial growth media. At the ultrastructural level, an increase in the periplasmalemmal space was observed due to the presence of a large number of vacuoles, together with a decrease in the relative volume of the nucleus when the cells were incubated in the presence of Cd. Pyrenoid and starch granules were observed and accumulation of spherical electron-dense bodies were also detected. X-ray spectra of these bodies for cells growing in artificial acid media showed intense C, O and Cd signals. In addition to these peaks, a strong Fe signal was also observed when cells were grown in natural acidic water.

ISOLATION AND CHARACTERIZATION OF A CELL WALL‐DEFECTIVE MUTANT OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA)1

Cell wall–defective strains of Chlamydomonas have played an important role in the development of transformation protocols for introducing exogenous DNA (foreign genes or cloned Chlamydomonas genes) into C. reinhardtii. To promote the development of similar protocols for transformation of the distantly related homothallic species, C. monoica, we used UV mutagenesis to obtain a mutant strain with a defective cell wall. The mutant, cw‐1, was first identified on the basis of irregular colony shape and was subsequently shown to have reduced plating efficiency and increased sensitivity to lysis by a non‐ionic detergent as compared with wild‐type cells. Tetrad analysis of crosses involving the cw‐1 mutant confirmed 2:2 segregation of the cw:cw+ phenotypes, indicating that the wall defect resulted from mutation of a single nuclear gene. The phenotype showed incomplete penetrance and variable expressivity. Although some cells had apparently normal cell walls as viewed by TEM, many cells of the cw‐1 strain had broken cell walls and others were protoplasts completely devoid of a cell wall. Several cw‐1 isolates obtained from crosses involving the original mutant strain showed a marked enhancement of the mutant phenotype and may prove especially useful for future work involving somatic cell fusions or development of transformation protocols.

Limiting steps of hydrogen production in Chlamydomonas reinhardtii and Synechocystis PCC 6803 as analysed by light-induced gas exchange transients

In the cyanobacterium Synechocystis PCC 6803 and in the microalga Chlamydomonas reinhardtii, transient hydrogen photo-production is observed when cells are exposed to light in anoxia. We measured changes in H 2, O 2, and CO 2 concentrations using time-resolved mass spectrometry in wild-type and mutant strains of Chlamydomonas and Synechocystis. In both organisms, non-photochemical reduction of the plastoquinone pool is shown to contribute to the initial H 2 photo-production. This pathway, which does not produce O 2, exhibits a low rate in normal conditions. From the effect of the uncoupler FCCP, we conclude that PS II-independent H 2 production in Chlamydomonas is limited by the trans-thylakoidal proton gradient. In Synechocystis, from the study of a mutant deficient in the NDH-1 complex (M55), we conclude that PS II-independent H 2 production is limited by recycling of NAD(P)H through the NDH-1 complex. Based on these conclusions, we propose strategies for optimising H 2 photo-production in these organisms.

Molecular Phylogeny and Taxonomic Revision of Chlamydomonas (Chlorophyta). I. Emendation of Chlamydomonas Ehrenberg and Chloromonas Gobi, and Description of Oogamochlamys gen. nov. and Lobochlamys gen. nov.

The genus Chlamydomonas (including Chloromonas) is one of the largest green algal genera comprising more than 600 species. To initiate a comprehensive analysis of the phylogeny and systematics of the genus, we determined nuclear-encoded SSU rRNA sequences from 32 strains of Chlamydomonas, Chloromonas and Chlorogonium with emphasis on oogamous taxa and related strains, and incorporated these into global molecular phylogenetic analyses of 132 strains of Chlorophyceae. In addition, we studied the morphology and reproduction of oogamous and related strains by light microscopy. We recognize and designate 18 monophyletic lineages (clades) within the Chlorophyceae, 11 of which are confined to the CW (basal bodies displaced clockwise) subgroup. The majority of clades recognized within the Chlorophyceae do not correspond to any of the traditional classification systems, which are still largely based on the organization level. Strains assigned to Chlamydomonas and Chloromonas were found in seven different clades confirming the polyphyly of the two genera as presently conceived. To initiate the taxonomic revision of Chlamydomonas, C. reinhardtii is proposed as the conserved type of the genus. In consequence, species in clades other than the clade containing C. reinhardtii must be transferred to other genera, a process initiated in this contribution. The oogamous strains studied represent a monophyletic lineage, which is described as Oogamochlamys gen. nov. comprising three species (O. gigantea, O. zimbabwiensis and O. ettlii spec. nov.). The sister clade to Oogamochlamys consists of isogamous strains characterized by chloroplasts with incisions and is described as Lobochlamys gen. nov. with two species (L. culleus and L. segnis). Another clade is characterized by asteroid or perforated, parietal chloroplasts and contains the type species of Chloromonas (C. reticulata). Thus, the polyphyletic Chloromonas (traditionally defined as "Chlamydomonas without pyrenoids") can be legitimized as a monophyletic genus by restriction to this clade and is here emended on the basis of chloroplast characters (the clade contains strains with or without pyrenoids thus rejecting the character "absence of pyrenoids").