A rapid method for chloroplast isolation from the green alga Chlamydomonas reinhardtii

This method has been developed to yield highly purified intact chloroplasts from Chlamydomonas reinhardtii. This procedure involves breaking cell-wall-deficient cells by passage through a narrow-bore syringe needle and purifying the intact chloroplasts by differential centrifugation and Percoll gradient centrifugation. This procedure can be completed in less than 3 h and is capable of generating relatively high yields of chloroplasts that should be useful for researchers studying the biochemistry and cell biology of C. reinhardtii chloroplasts.     

Crystal structure of chloroplast fructose-1,6-bisphosphate aldolase from the green alga Chlamydomonas reinhardtii

The Calvin-Benson cycle fixes carbon dioxide into organic triosephosphates through the collective action of eleven conserved enzymes. Regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco-mediated carboxylation, requires two lyase reactions catalyzed by fructose-1,6-bisphosphate aldolase (FBA). While cytoplasmic FBA has been extensively studied in non-photosynthetic organisms, functional and structural details are limited for chloroplast FBA encoded by oxygenic phototrophs. Here we determined the crystal structure of plastidial FBA from the unicellular green alga Chlamydomonas reinhardtii (Cr). We confirm that CrFBA folds as a TIM barrel, describe its catalytic pocket and homo-tetrameric state. Multiple sequence profiling classified the photosynthetic paralogs of FBA in a distinct group from non-photosynthetic paralogs. We mapped the sites of thiol- and phospho-based post-translational modifications known from photosynthetic organisms and predict their effects on enzyme catalysis.     

Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii

There is a growing interest in the use of microalgae as low‐cost hosts for the synthesis of recombinant products such as therapeutic proteins and bioactive metabolites. In particular, the chloroplast, with its small, genetically tractable genome (plastome) and elaborate metabolism, represents an attractive platform for genetic engineering. In Chlamydomonas reinhardtii, none of the 69 protein‐coding genes in the plastome uses the stop codon UGA, therefore this spare codon can be exploited as a useful synthetic biology tool. Here, we report the assignment of the codon to one for tryptophan and show that this can be used as an effective strategy for addressing a key problem in chloroplast engineering: namely, the assembly of expression cassettes in Escherichia coli when the gene product is toxic to the bacterium. This problem arises because the prokaryotic nature of chloroplast promoters and ribosome‐binding sites used in such cassettes often results in transgene expression in E. coli, and is a potential issue when cloning genes for metabolic enzymes, antibacterial proteins and integral membrane proteins. We show that replacement of tryptophan codons with the spare codon (UGG→UGA) within a transgene prevents functional expression in E. coli and in the chloroplast, and that co‐introduction of a plastidial trnW gene carrying a modified anticodon restores function only in the latter by allowing UGA readthrough. We demonstrate the utility of this system by expressing two genes known to be highly toxic to E. coli and discuss its value in providing an enhanced level of biocontainment for transplastomic microalgae.     

Regulation of molybdenum cofactor species in the green alga Chlamydomonas reinhardtii

Molybdenum cofactor (MoCo) of molybdoenzymes is constitutively produced in cells of the green alga Chlamydomonas reinhardtii grown in ammonium media, under which conditions certain molybdoenzymes are not synthesized. In soluble form, MoCo was found to be present in several forms: (i) as a low Mr free species; (ii) bound to a MoCo-carrier protein of about 50 kDa that could release MoCo to directly reconstitute in vitro nitrate reductase activity in the nit-1 mutant of Neurospora crassa, but not to Thiol-Sepharose which, in contrast, bonded free MoCo; and (iii) bound to other proteins, putatively constitutive molybdoenzymes, which only released MoCo after a denaturing treatment. The amount of total MoCo (free, carrier-bound and heat releasable forms) was dependent on the growth phase of cell cultures. Constitutive levels of total MoCo in ammonium-grown cells markedly increased when cells were transferred to media lacking ammonium (nitrate, urea or nitrogen-free media). This increase did not require de novo protein synthesis and was stimulated by light. Levels of both total MoCo and free plus carrier-bound MoCo seemed to be unrelated to either nitrate reductase synthesis or functioning of nit-1 and nit-2 genes responsible for nitrate reductase structure and regulation, respectively. Results suggest that MoCo is continuously synthesized in C. reinhardtii and that its levels are regulated by ammonium in a way independent of nitrate reductase synthesis.     

In vivo localization of centrin in the green alga Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii has been used as a model system to study flagellar assembly, centriole assembly, and cell cycle events. These processes are dynamic. Therefore, protein targeting and protein-protein interactions should be evaluated in vivo. To be able to study dynamic processes in C. reinhardtii in vivo, we have explored the use of the green fluorescent protein (GFP). A construct containing a fusion of centrin and GFP was incorporated into the genome as a single copy. The selected clone shows expression in 25-50% of the cells. Centrin-GFP was targeted in vivo to the nuclear basal body connectors and the distal connecting fibers. At the electron microscopic level, it was also localized to the flagellar transitional regions. EM data of transformants indicate that there are some abnormalities in the centrin-containing structures. The transitional region consists of only the transverse septum or has lesions in the H-piece. The distal connecting fibers are thinner and their characteristic crossbands seem to be incomplete. Deflagellation is not affected since more than 95% of the cells deflagellate. Also basal body segregation is not affected since cells with an abnormal flagellar number were not detected. Functional studies of the centrin-GFP fusion show the characteristic calcium-induced mobility shift in SDS-PAGE. Immunofluorescence revealed that during cell division, centrin-GFP remains associated with the basal bodies. In vivo localization of the fusion protein during cell division shows that in metaphase centrin-GFP appears as two opposing spots located close to the spindle poles. The distance between the spots increases as the cells progress through anaphase and then decreases during telophase. GFP is a useful tool to study dynamic processes in the cytoskeleton of C. reinhardtii.     

Novel shuttle markers for nuclear transformation of the green alga Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii today is a premier model organism for the study of green algae and plants. Yet the efficient engineering of its nuclear genome requires development of new antibiotic resistance markers. We have recoded, based on codon usage in the nuclear genome, the AadA marker that has been used previously for chloroplast transformation. The recoded AadA gene, placed under the control of the HSP70A-RBCS2 hybrid promoter and preceded by the RbcS2 chloroplast-targeting peptide, can be integrated into the nuclear genome by electroporation, conferring resistance to spectinomycin and streptomycin. Transformation efficiency is markedly increased when vector sequences are completely eliminated from the transforming DNA. Antibiotic resistance is stable for several months in the absence of selection pressure. Shuttle markers allowing selection in both Chlamydomonas and Escherichia coli would also be a useful asset. By placing an artificial bacterial promoter and Shine-Dalgarno sequence in frame within the AadA coding sequence, we generated such a shuttle marker. To our surprise, we found that the classical AphVIII construct already functions as a shuttle marker. Finally, we developed a method to introduce the AadA and AphVIII markers into the vector part of the bacterial artificial chromosomes (BACs) of the Chlamydomonas genomic DNA library. Our aim was to facilitate complementation studies whenever the test gene cannot be selected for directly. After transformation of a petC mutant with a modified BAC carrying the AphVIII marker along with the PETC gene in the insert, almost half of the paromomycin-resistant transformants obtained showed restoration of phototrophy, indicating successful integration of the unselected test gene. With AadA, cotransformation was also observed, but with a lower efficiency.     

Carotenoid deficiency triggers autophagy in the model green alga Chlamydomonas reinhardtii

All aerobic organisms have developed sophisticated mechanisms to prevent, detect and respond to cell damage caused by the unavoidable production of reactive oxygen species (ROS). Plants and algae are able to synthesize specific pigments in the chloroplast called carotenoids to prevent photo-oxidative damage caused by highly reactive by-products of photosynthesis. In this study we used the unicellular green alga Chlamydomonas reinhardtii to demonstrate that defects in carotenoid biosynthesis lead to the activation of autophagy, a membrane-trafficking process that participates in the recycling and degradation of damaged or toxic cellular components. Carotenoid depletion caused by either the mutation of phytoene synthase or the inhibition of phytoene desaturase by the herbicide norflurazon, resulted in a strong induction of autophagy. We found that high light transiently activates autophagy in wild-type Chlamydomonas cells as part of an adaptation response to this stress. Our results showed that a Chlamydomonas mutant defective in the synthesis of specific carotenoids that accumulate during high light stress exhibits constitutive autophagy. Moreover, inhibition of the ROS-generating NADPH oxidase partially reduced the autophagy induction associated to carotenoid deficiency, which revealed a link between photo-oxidative damage, ROS accumulation and autophagy activation in Chlamydomonas cells with a reduced carotenoid content.     

Carotenoid deficiency triggers autophagy in the model green alga Chlamydomonas reinhardtii

All aerobic organisms have developed sophisticated mechanisms to prevent, detect and respond to cell damage caused by the unavoidable production of reactive oxygen species (ROS). Plants and algae are able to synthesize specific pigments in the chloroplast called carotenoids to prevent photo-oxidative damage caused by highly reactive by-products of photosynthesis. In this study we used the unicellular green alga Chlamydomonas reinhardtii to demonstrate that defects in carotenoid biosynthesis lead to the activation of autophagy, a membrane-trafficking process that participates in the recycling and degradation of damaged or toxic cellular components. Carotenoid depletion caused by either the mutation of phytoene synthase or the inhibition of phytoene desaturase by the herbicide norflurazon, resulted in a strong induction of autophagy. We found that high light transiently activates autophagy in wild-type Chlamydomonas cells as part of an adaptation response to this stress. Our results showed that a Chlamydomonas mutant defective in the synthesis of specific carotenoids that accumulate during high light stress exhibits constitutive autophagy. Moreover, inhibition of the ROS-generating NADPH oxidase partially reduced the autophagy induction associated to carotenoid deficiency, which revealed a link between photo-oxidative damage, ROS accumulation and autophagy activation in Chlamydomonas cells with a reduced carotenoid content.     

Analysis of lysine-rich histones from the unicellular green alga Chlamydomonas reinhardtii

The H1 histones of the unicellular green alga Chlamydomonas reinhardtii were extracted from isolated nuclei, fractionated by high performance liquid chromatography, and analyzed by two-dimensional electrophoresis, peptide mapping, and N-terminal sequencing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 5% perchloric acid extracts of isolated C. reinhardtii nuclei revealed two H1 proteins (H1A and H1B). Two-dimensional gel analysis did not reveal heterogeneity of either algal H1 protein, but did detect differences in the hydrophobic amino acid content of the C. reinhardtii H1A and H1B. Digestion of H1A and H1B with V8 protease revealed two distinctly different peptide maps. C. reinhardtii H1 peptide maps were not at all similar to those of Pisum H1, but algal and pea H2B peptide maps did show some peptides in common. Seventeen amino acid residues were obtained from C. reinhardtii H1A amino terminal sequencing, while the H1B N-terminus was blocked. A search of protein data bases revealed no sequence homology of the H1A N-terminus with any known protein. Chlamydomonas histones fractionated by high performance liquid chromatography revealed minor components (histone variants) for H2A and H2B. The amino acid composition of Chlamydomonas lysine-rich histones was compared to those of various other unicellular algae.     

NMR structures of thioredoxin m from the green alga Chlamydomonas reinhardtii

Chloroplast thioredoxin m from the green alga Chlamydomomas reinhardtii is very efficiently reduced in vitro and in vivo in the presence of photoreduced ferredoxin and a ferredoxin dependent ferredoxin-thioredoxin reductase. Once reduced, thioredoxin m has the capability to quickly activate the NADP malate dehydrogenase (EC 1.1.1.82) a regulatory enzyme involved in an energy-dependent assimilation of carbon dioxide in C4 plants. This activation is the result of the reduction of two disulfide bridges by thioredoxin m, that are located at the N- and C-terminii of the NADP malate dehydrogenase. The molecular structure of thioredoxin m was solved using NMR and compared to other known thioredoxins. Thioredoxin m belongs to the prokaryotic type of thioredoxin, which is divergent from the eukaryotic-type thioredoxins also represented in plants by the h (cytosolic) and f (chloroplastic) types of thioredoxins. The dynamics of the molecule have been assessed using (15)N relaxation data and are found to correlate well with regions of disorder found in the calculated NMR ensemble. The results obtained provide a novel basis to interpret the thioredoxin dependence of the activation of chloroplast NADP-malate dehydrogenase. The specific catalytic mechanism that takes place in the active site of thioredoxins is also discussed on the basis of the recent new understanding and especially in the light of the dual general acid-base catalysis exerted on the two cysteines of the redox active site. It is proposed that the two cysteines of the redox active site may insulate each other from solvent attack by specific packing of invariable hydrophobic amino acids.     

The ferredoxin-thioredoxin system of a green alga,Chlamydomonas reinhardtii

The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher-plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (M_rs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11 500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa (similar) subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa (variable) sub-unit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.Abbreviations DEAE diethylaminoethyl - ELISA enzyme-linked immunosorption assay - FBPase fructose 1,6-bisphosphatase - Fd ferredoxin - FPLC fast protein liquid chromatography - FTR ferredoxin-thioredoxin reductase - FT system ferredoxin-thioredoxin system - kDa kilodaltons - M_r relative molecular mass - NADP-MDH NADP-malate dehydrogenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported in part by a grant from the National Aeronautics and Space Administration. We would like to thank Don Carlson and Jacqueline Girard for their assistance with cell cultures.     

Architecture and evolution of subtelomeres in the unicellular green alga Chlamydomonas reinhardtii

In most eukaryotes, subtelomeres are dynamic genomic regions populated by multi-copy sequences of different origins, which can promote segmental duplications and chromosomal rearrangements. However, their repetitive nature has complicated the efforts to sequence them, analyse their structure and infer how they evolved. Here, we use recent genome assemblies of Chlamydomonas reinhardtii based on long-read sequencing to comprehensively describe the subtelomere architecture of the 17 chromosomes of this model unicellular green alga. We identify three main repeated elements present at subtelomeres, which we call Sultan, Subtile and Suber, alongside three chromosome extremities with ribosomal DNA as the only identified component of their subtelomeres. The most common architecture, present in 27 out of 34 subtelomeres, is a heterochromatic array of Sultan elements adjacent to the telomere, followed by a transcribed Spacer sequence, a G-rich microsatellite and transposable elements. Sequence similarity analyses suggest that Sultan elements underwent segmental duplications within each subtelomere and rearranged between subtelomeres at a much lower frequency. Analysis of other green algae reveals species-specific repeated elements that are shared across subtelomeres, with an overall organization similar to C. reinhardtii. This work uncovers the complexity and evolution of subtelomere architecture in green algae.