Unicellular Green Alga Chlamydomonas Reinhardtii Research Articles

Characterization of Chlamydomonas reinhardtii Mutants That Exhibit Strong Positive Phototaxis.

The most motile phototrophic organisms exhibit photo-induced behavioral responses (photobehavior) to inhabit better light conditions for photosynthesis. The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to study photobehavior. Several years ago, we found that C. reinhardtii cells reverse their phototactic signs (i.e., positive and negative phototaxis) depending on the amount of reactive oxygen species (ROS) accumulated in the cell. However, its molecular mechanism is unclear. In this study, we isolated seven mutants showing positive phototaxis, even after the induction of negative phototaxis (ap1~7: always positive) to understand the ROS-dependent regulatory mechanism for the phototactic sign. We found no common feature in the mutants regarding their growth, high-light tolerance, and photosynthetic phenotypes. Interestingly, five of them grew faster than the wild type. These data suggest that the ROS-dependent regulation of the phototactic sign is not a single pathway and is affected by various cellular factors. Additionally, the isolation and analyses of mutants with defects in phototactic-sign regulation may provide clues for their application to the efficient cultivation of algae.

A FORWARD GENETIC SCREEN FOR ISOLATION OF FUSION DEFECTIVE MATING TYPE MINUS MUTANTS IN CHLAMYDOMONAS REINHARDTII

Many biological processes require cell fusion, therefore defects in cell fusion result in many diseases. The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to study cell-cell fusion. The objective of this study was to identify genes that are involved in C. reinhardtii mating type minus (MT_) gamete fusion. A forward genetics approach was taken in our work. We created several MT_ fusion defective mutants using DNA insertional mutagenesis. These mutants were normal in the early stages of mating; they agglutinated with mating type plus (MT+), removed their walls, adhered to their mating partner through their mating structures, but the cells did not fuse, indicating that the DNA insertional mutants were defective in the latest stages of fusion. The number of insertions was confirmed by Southern blots. Mutant J1 had one insertion and the flanking genomic DNA was cloned by TAIL-PCR and RESDA-PCR. The insertion is in a gene predicted to be involved in 5-deoxystrigol biosynthesis.

A Lipid Bodies-Associated Galactosyl Hydrolase Is Involved in Triacylglycerol Biosynthesis and Galactolipid Turnover in the Unicellular Green Alga Chlamydomonas reinhardtii.

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the main constituent lipids of thylakoid and chloroplast envelop membranes. Many microalgae can accumulate large amounts of triacylglycerols (TAGs) under adverse environmental conditions, which is accompanied by degradation of the photosynthetic membrane lipids. However, the process mediating the conversion from galactolipids to TAG remains largely unknown. In this study, we performed genetic and biochemical analyses of galactosyl hydrolases (CrGH) identified in the proteome of lipid bodies of the green microalga Chlamydomonas reinhardtii. The recombinant CrGH was confirmed to possess galactosyl hydrolase activity by using o-nitrophenyl-β-D-galactoside as the substrate, and the Michaelis constant (Km) and Kcat of CrGH were 13.98 μM and 3.62 s−1, respectively. Comparative lipidomic analyses showed that the content of MGDG and DGDG increased by 14.42% and 24.88%, respectively, in the CrGH-deficient mutant as compared with that of the wild type cc4533 grown under high light stress conditions, and meanwhile, the TAG content decreased by 32.20%. Up-regulation of CrGH at both a gene expression and protein level was observed under high light stress (HL) conditions. In addition, CrGH was detected in multiple subcellular localizations, including the chloroplast envelope, mitochondria, and endoplasmic reticulum membranes. This study uncovered a new paradigm mediated by the multi-localized CrGH for the conversion of the photosynthetic membranes to TAGs.

Co-expression networks in Chlamydomonas reveal significant rhythmicity in batch cultures and empower gene function discovery.

The unicellular green alga Chlamydomonas reinhardtii is a choice reference system for the study of photosynthesis and chloroplast metabolism, cilium assembly and function, lipid and starch metabolism, and metal homeostasis. Despite decades of research, the functions of thousands of genes remain largely unknown, and new approaches are needed to categorically assign genes to cellular pathways. Growing collections of transcriptome and proteome data now allow a systematic approach based on integrative co-expression analysis. We used a dataset comprising 518 deep transcriptome samples derived from 58 independent experiments to identify potential co-expression relationships between genes. We visualized co-expression potential with the R package corrplot, to easily assess co-expression and anti-correlation between genes. We extracted several hundred high-confidence genes at the intersection of multiple curated lists involved in cilia, cell division, and photosynthesis, illustrating the power of our method. Surprisingly, Chlamydomonas experiments retained a significant rhythmic component across the transcriptome, suggesting an underappreciated variable during sample collection, even in samples collected in constant light. Our results therefore document substantial residual synchronization in batch cultures, contrary to assumptions of asynchrony. We provide step-by-step protocols for the analysis of co-expression across transcriptome data sets from Chlamydomonas and other species to help foster gene function discovery.

Regulated Chloroplast Gene Expression in Chlamydomonas.

The green unicellular alga Chlamydomonas reinhardtii has emerged as a very attractive model system for chloroplast genetic engineering. Algae can be transformed readily at the chloroplast level through bombardment of cells with a gene gun and transformants can be selected using antibiotic resistance or phototrophic growth. An inducible chloroplast gene expression system could be very useful for several reasons. First, it could be used to elucidate the function of essential chloroplast genes required for cell growth and survival. Second, it could be very helpful for expressing proteins which are toxic to the algal cells. Third, it would allow for the reversible depletion of photosynthetic complexes, thus making it possible to study their biogenesis in a controlled fashion. Fourth, it opens promising possibilities for hydrogen production in Chlamydomonas. Here we describe an inducible/ repressible chloroplast gene expression system in Chlamydomonas in which the copper-regulated Cyc6 promoter or the vitamin-controlled MetE promoter and TPP riboswitch drive the expression of the nuclear Nac2 gene encoding a protein which is targeted to the chloroplast where it acts specifically on the chloroplast psbD 5' untranslated region and is required for the stable accumulation of the psbD mRNA and photosystem II. The system can be used for any chloroplast gene or trans-gene by placing it under the control of the psbD 5'untranslated region.

Water oxidation by photosystem II is the primary source of electrons for sustained H2 photoproduction in nutrient-replete green algae

The unicellular green alga Chlamydomonas reinhardtii is capable of photosynthetic H2 production. H2 evolution occurs under anaerobic conditions and is difficult to sustain due to 1) competition between [FeFe]-hydrogenase (H2ase), the key enzyme responsible for H2 metabolism in algae, and the Calvin-Benson-Bassham (CBB) cycle for photosynthetic reductants and 2) inactivation of H2ase by O2 coevolved in photosynthesis. Recently, we achieved sustainable H2 photoproduction by shifting algae from continuous illumination to a train of short (1 s) light pulses, interrupted by longer (9 s) dark periods. This illumination regime prevents activation of the CBB cycle and redirects photosynthetic electrons to H2ase. Employing membrane-inlet mass spectrometry and [Formula: see text], we now present clear evidence that efficient H2 photoproduction in pulse-illuminated algae depends primarily on direct water biophotolysis, where water oxidation at the donor side of photosystem II (PSII) provides electrons for the reduction of protons by H2ase downstream of photosystem I. This occurs exclusively in the absence of CO2 fixation, while with the activation of the CBB cycle by longer (8 s) light pulses the H2 photoproduction ceases and instead a slow overall H2 uptake is observed. We also demonstrate that the loss of PSII activity in DCMU-treated algae or in PSII-deficient mutant cells can be partly compensated for by the indirect (PSII-independent) H2 photoproduction pathway, but only for a short (<1 h) period. Thus, PSII activity is indispensable for a sustained process, where it is responsible for more than 92% of the final H2 yield.

Cold Stress Response: An Overview in Chlamydomonas.

Low temperature (or cold) is one of the major environmental factors that limit the growth and development of many plants. Various plant species have evolved complex mechanisms to adjust to decreased temperature. Mesophilic chlorophytes are a widely distributed group of eukaryotic photosynthetic organisms, but there is insufficient information about the key molecular processes of their cold acclimation. The best available model for studying how chlorophytes respond to and cope with variations in temperature is the unicellular green alga Chlamydomonas reinhardtii. Chlamydomonas has been widely used for decades as a model system for studying the fundamental mechanisms of the plant heat stress response. At present, unraveling novel cold-regulated events in Chlamydomonas has attracted increasing research attention. This mini-review summarizes recent progress on low-temperature-dependent processes in the model alga, while information on other photosynthetic organisms (cyanobacteria and land plants) was used to strengthen generalizations or specializations of cold-induced mechanisms in plant evolution. Here, we describe recent advances in our understanding of cold stress response in Chlamydomonas, discuss areas of controversy, and highlight potential future directions in cold acclimation research.

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.

Overexpression of Sedoheptulose-1,7-Bisphosphatase Enhances Photosynthesis in Chlamydomonas reinhardtii and Has No Effect on the Abundance of Other Calvin-Benson Cycle Enzymes.

The productivity of plants and microalgae needs to be increased to feed the growing world population and to promote the development of a low-carbon economy. This goal can be achieved by improving photosynthesis via genetic engineering. In this study, we have employed the Modular Cloning strategy to overexpress the Calvin-Benson cycle (CBC) enzyme sedoheptulose-1,7-bisphosphatase (SBP1) up to threefold in the unicellular green alga Chlamydomonas reinhardtii. The protein derived from the nuclear transgene represented ∼0.3% of total cell protein. Photosynthetic rate and growth were significantly increased in SBP1-overexpressing lines under high-light and elevated CO2 conditions. Absolute quantification of the abundance of all other CBC enzymes by the QconCAT approach revealed no consistent differences between SBP1-overexpressing lines and the recipient strain. This analysis also revealed that the 11 CBC enzymes represent 11.9% of total cell protein in Chlamydomonas. Here, the range of concentrations of CBC enzymes turned out to be much larger than estimated earlier, with a 128-fold difference between the most abundant CBC protein (rbcL) and the least abundant (triose phosphate isomerase). Accordingly, the concentrations of the CBC intermediates are often but not always higher than the binding site concentrations of the enzymes for which they act as substrates. The enzymes with highest substrate to binding site ratios might represent good candidates for overexpression in subsequent engineering steps.

Evolutionary diverse Chlamydomonas reinhardtii Old Yellow Enzymes reveal distinctive catalytic properties and potential for whole-cell biotransformations

Old Yellow Enzymes (OYEs) selectively reduce carbon‑carbon double bonds of a broad range of substrates with excellent stereoselectivity. Current challenges of their application in bio-based industrial processes are the cost-efficient regeneration of the nicotinamide co-substrate and expanding the range of industrially relevant substrates. Microalgae represent a rich and mostly untapped source for OYEs with novel catalytic properties and are promising whole-cell factories that regenerate nicotinamide by photosynthesis. By comparative phylogenetics, we identified 20 putative OYEs in eleven algal species. Three recombinant OYEs from the unicellular green alga Chlamydomonas reinhardtii (CrOYEs) reveal diverse biocatalytic properties and potential for enzyme optimization. High substrate conversion activities of living algal wild type and mutant cells linked the biocatalytic profiles to in vivo functionality and demonstrate the potential of microalgae as sources for distinctive OYE activities and for light-driven biotransformations.

The pyrenoid.

Wang and Jonikas take a look at an unconventional organelle, the pyrenoid.

Distinctive structural properties of THB11, a pentacoordinate Chlamydomonas reinhardtii truncated hemoglobin with N- and C-terminal extensions

Hemoglobins (Hbs) utilize heme b as a cofactor and are found in all kingdoms of life. The current knowledge reveals an enormous variability of Hb primary sequences, resulting in topological, biochemical and physiological individuality. As Hbs appear to modulate their reactivities through specific combinations of structural features, predicting the characteristics of a given Hb is still hardly possible. The unicellular green alga Chlamydomonas reinhardtii contains 12 genes encoding diverse Hbs of the truncated lineage, several of which possess extended N- or C-termini of unknown function. Studies on some of the Chlamydomonas Hbs revealed yet unpredictable structural and biochemical variations, which, along with a different expression of their genes, suggest diverse physiological roles. Chlamydomonas thus represents a promising system to analyze the diversification of Hb structure, biochemistry and physiology. Here, we report the crystal structure, resolved to 1.75 Å, of the heme-binding domain of cyanomet THB11 (Cre16.g662750), one of the pentacoordinate algal Hbs, which offer a free Fe-coordination site in the reduced state. The overall fold of THB11 is conserved, but individual features such as a kink in helix E, a tilted heme plane and a clustering of methionine residues at a putative tunnel exit appear to be unique. Both N- and C-termini promote the formation of oligomer mixtures, and the absence of the C terminus results in reduced nitrite reduction rates. This work widens the structural and biochemical knowledge on the 2/2Hb family and suggests that the N- and C-terminal extensions of the Chlamydomonas 2/2Hbs modulate their reactivity by intermolecular interactions.

Microalgal plastidial lysophosphatidic acid acyltransferase interacts with upstream glycerol-3-phosphate acyltransferase and defines its substrate selectivity via the two transmembrane domains

The plastidial lysophosphatidic acid acyltransferase of the unicellular green alga Chlamydomonas reinhardtii (CrLPAAT1) is a key enzyme involved in triacylglycerol biosynthesis. However, research on the biochemical characteristics of CrLPAAT1 has been impeded by its membrane-bound nature. In this study, the recombinant CrLPAAT1 was purified in a soluble form and was utilized for comprehensive biochemical characterization. The recombinant CrLPAAT1 favors the conditions of pH 6.5–7.5, 30 °C and the presence of magnesium ion in vitro. Similar with the CrLPAAT1 associated with the membranes, the purified CrLPAAT1 prefers to utilize C16:0-CoA over other acyl donors, whereas it showed broader substrate selectivity than the membrane-bound enzyme. By comparing the wild-type CrLPAAT1 and a transmembrane domain-truncated enzyme, it was uncovered in this study that the two transmembrane domains of CrLPAAT1 are involved in shaping its substrate preference for C16:0-CoA. Additionally, CrLPAAT1 was found to be interacting with the water-soluble plastidial glycerol-3-phosphate acyltransferase (CrGAPTcl) via its two transmembrane domains in vitro. The interaction between CrLPAAT1 and CrGPATcl can be negatively regulated by both the acyl-CoAs and lysophosphatidic acid in a dosage-dependent manner. Such a regulation pattern may represent a novel mechanism adopted by algal cells to control lipid metabolism homeostasis under various environmental conditions.

Unraveling enhanced membrane lipid biosynthesis in Chlamydomonas reinhardtii starchless mutant sta6 by using an electrospray ionization mass spectrometry-based lipidomics method

The unicellular green alga Chlamydomonas reinhardtii, a well-established model organism, has been widely used in dissecting glycerolipid metabolism in oxygenating photosynthetic organisms. In previous studies, it has been found that shunting carbon precursors from the starch synthesis pathway can lead to a 10-fold increase in TAG content as compared to the wild type, but it is unknown whether inactivation of AGPase may affect membrane lipids biosynthesis. The study aims to investigate global changes in lipid metabolism and homeostasis in the starchless mutant C. reinhardtii sta6. By utilizing an electrospray ionization/mass spectrometry (ESI/MS)-based lipidomics approach, a total of 105 membrane lipid molecules of C. reinhardtii were resolved, including 16 monogalactosyldiacylglycerol (MGDG), 16 digalactosyldiacylglycerol (DGDG), 11 phosphatidylglycerol (PG), 6 sulfoquinovosyldiacylglycerol (SQDG), 49 diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), 2 phosphatidylethanolamine (PE), and 5 phosphatidylinositol (PI) molecules. The quantitative results indicated that the membrane lipid profiles were similar between the two C. reinhardtii strains grown under both low- and high-light conditions, but the cellular contents of a great number of lipids were altered in sta6 due to the defect in starch biosynthesis. Under low-light conditions, sta6 accumulated more PI, MGDG, DGDG but less amounts of DGTS as compared to WT. Under high light, sta6 cells contained higher content membrane lipids than cc-124, except for PG, which is more or less similar in both strains. Our results demonstrate that the cellular membrane lipid homeostasis underwent profound changes in the starchless mutant, and thereby its physiological impact remains to be explored.

When Unity Is Strength: The Strategies Used by Chlamydomonas to Survive Environmental Stresses.

The unicellular green alga Chlamydomonas reinhardtii is a valuable model system to study a wide spectrum of scientific fields, including responses to environmental conditions. Most studies are performed under optimal growth conditions or under mild stress. However, when environmental conditions become harsher, the behavior of this unicellular alga is less well known. In this review we will show that despite being a unicellular organism, Chlamydomonas can survive very severe environmental conditions. To do so, and depending on the intensity of the stress, the strategies used by Chlamydomonas can range from acclimation to the formation of multicellular structures, or involve programmed cell death.

Distinct roles of Argonaute in the green alga Chlamydomonas reveal evolutionary conserved mode of miRNA-mediated gene expression

The unicellular green alga Chlamydomonas reinhardtii is evolutionarily divergent from higher plants, but has a fully functional silencing machinery including microRNA (miRNA)-mediated translation repression and mRNA turnover. However, distinct from the metazoan machinery, repression of gene expression is primarily associated with target sites within coding sequences instead of 3′UTRs. This feature indicates that the miRNA-Argonaute (AGO) machinery is ancient and the primary function is for post transcriptional gene repression and intermediate between the mechanisms in the rest of the plant and animal kingdoms. Here, we characterize AGO2 and 3 in Chlamydomonas, and show that cytoplasmically enriched Cr-AGO3 is responsible for endogenous miRNA-mediated gene repression. Under steady state, mid-log phase conditions, Cr-AGO3 binds predominantly miR-C89, which we previously identified as the predominant miRNA with effects on both translation repression and mRNA turnover. In contrast, the paralogue Cr-AGO2 is nuclear enriched and exclusively binds to 21-nt siRNAs. Further analysis of the highly similar Cr-AGO2 and Cr-AGO 3 sequences (90% amino acid identity) revealed a glycine-arginine rich N-terminal extension of ~100 amino acids that, given previous work on unicellular protists, may associate AGO with the translation machinery. Phylogenetic analysis revealed that this glycine-arginine rich N-terminal extension is present outside the animal kingdom and is highly conserved, consistent with our previous proposal that miRNA-mediated CDS-targeting operates in this green alga.

Partially Redundant Actin Genes in Chlamydomonas Control Transition Zone Organization and Flagellum-Directed Traffic

SUMMARYThe unicellular green alga Chlamydomonas reinhardtii is a biflagellated cell with two actin genes: one encoding a conventional actin (IDA5) and the other encoding a divergent novel actin-like protein (NAP1). Here, we probe how actin redundancy contributes to flagellar assembly. Disrupting a single actin allows complete flagellar assembly. However, when disrupting both actins using latrunculin B (LatB) treatment on the nap1 mutant background, we find that actins are necessary for flagellar growth from newly synthesized limiting flagellar proteins. Under total actin disruption, transmission electron microscopy identified an accumulation of Golgi-adjacent vesicles. We also find that there is a mislocalization of a key transition zone gating and ciliopathy protein, NPHP-4. Our experiments demonstrate that each stage of flagellar biogenesis requires redundant actin function to varying degrees, with an absolute requirement for these actins in transport of Golgi-adjacent vesicles and flagellar incorporation of newly synthesized proteins.

Construction of a novel vector for the nuclear transformation of the unicellular green alga Chlamydomonas reinhardtii and its stable expression

Plasmid pBI221aadAGUS which carried both of GUS (β-glucuronidase) and aadA (aminoglyoside transferase) genes besides of the 35S cauliflower mosaic virus promoter was constructed and used for stable nuclear transformation of Chlamydomonas reinhardtii. The vector was transformed into the alga by particle gun bombardment and two positive colonies were selected on spectinomycin–containing medium. The restriction analysis of the DNA of the positive colonies showed that aadA was inserted in two orientations. The presence of introduced genes in the transformed colonies was confirmed by (PCR) using primers specific to GUS and aadA genes. The expression of aadA and GUS genes was revealed in all colonies that were grown on spectinomycin in liquid culture for 3–4 generations. The usefulness of this vector, differing in the orientation of the aadA cassette, was manisfested by transforming C. reinhardtii to spectinomycin resistance in the stable expression. This constructed plasmid-based expression vector system would help to unravel the functions of various genes in the green alga.

De novo origins of multicellularity in response to predation

The transition from unicellular to multicellular life was one of a few major events in the history of life that created new opportunities for more complex biological systems to evolve. Predation is hypothesized as one selective pressure that may have driven the evolution of multicellularity. Here we show that de novo origins of simple multicellularity can evolve in response to predation. We subjected outcrossed populations of the unicellular green alga Chlamydomonas reinhardtii to selection by the filter-feeding predator Paramecium tetraurelia. Two of five experimental populations evolved multicellular structures not observed in unselected control populations within ~750 asexual generations. Considerable variation exists in the evolved multicellular life cycles, with both cell number and propagule size varying among isolates. Survival assays show that evolved multicellular traits provide effective protection against predation. These results support the hypothesis that selection imposed by predators may have played a role in some origins of multicellularity.

Multiomics resolution of molecular events during a day in the life of Chlamydomonas

The unicellular green alga Chlamydomonas reinhardtii displays metabolic flexibility in response to a changing environment. We analyzed expression patterns of its three genomes in cells grown under light-dark cycles. Nearly 85% of transcribed genes show differential expression, with different sets of transcripts being up-regulated over the course of the day to coordinate cellular growth before undergoing cell division. Parallel measurements of select metabolites and pigments, physiological parameters, and a subset of proteins allow us to infer metabolic events and to evaluate the impact of the transcriptome on the proteome. Among the findings are the observations that Chlamydomonas exhibits lower respiratory activity at night compared with the day; multiple fermentation pathways, some oxygen-sensitive, are expressed at night in aerated cultures; we propose that the ferredoxin, FDX9, is potentially the electron donor to hydrogenases. The light stress-responsive genes PSBS, LHCSR1, and LHCSR3 show an acute response to lights-on at dawn under abrupt dark-to-light transitions, while LHCSR3 genes also exhibit a later, second burst in expression in the middle of the day dependent on light intensity. Each response to light (acute and sustained) can be selectively activated under specific conditions. Our expression dataset, complemented with coexpression networks and metabolite profiling, should constitute an excellent resource for the algal and plant communities.