Unicellular Green Alga Chlamydomonas Reinhardtii Research Articles

Analysis of Unique Motility of the Unicellular Green Alga Chlamydomonas reinhardtii at Low Temperatures down to -8 °C.

Previous studies of motility at low temperatures in Chlamydomonas reinhardtii have been conducted at temperatures of up to 15 °C. In this study, we report that C. reinhardtii exhibits unique motility at a lower temperature range (-8.7 to 1.7 °C). Cell motility was recorded using four low-cost, easy-to-operate observation systems. Fast Fourier transform (FFT) analysis at room temperature (20-27 °C) showed that the main peak frequency of oscillations ranged from 44 to 61 Hz, which is consistent with the 60 Hz beat frequency of flagella. At lower temperatures, swimming velocity decreased with decreasing temperature. The results of the FFT analysis showed that the major peak shifted to the 5-18 Hz range, suggesting that the flagellar beat frequency was decreasing. The FFT spectra had distinct major peaks in both temperature ranges, indicating that the oscillations were regular. This was not affected by the wavelength of the observation light source (white, red, green or blue LED) or the environmental spatial scale of the cells. In contrast, cells in a highly viscous (3.5 mPa·s) culture at room temperature showed numerous peaks in the 0-200 Hz frequency band, indicating that the oscillations were irregular. These findings contribute to a better understanding of motility under lower-temperature conditions in C. reinhardtii.

GATA family transcription factors in alga Chlamydomonas reinhardtii.

GATA family transcription factors (GATA-TFs) are metalloproteins that regulate many metabolic pathways. These conserved proteins recognize the consensus sequence (A/T)GATA(A/G) in the promoter regions of many genes and regulate their transcription in response to environmental signals. Currently, the study of GATA-TFs is of increasing interest. GATA genes and their proteins are most actively studied in vascular plants and fungi. Based on the results of numerous studies, it has been shown that GATA factors regulate the metabolic pathways of nitrogen and carbon, and also play a major role in the processes induced by light and circadian rhythms. In algae, GATA-TFs remain poorly studied, and information about them is scattered. In this work, all known data on GATA-TFs in the unicellular green alga Chlamydomonas reinhardtii has been collected and systematized. The genome of this alga contains 12 GATA coding genes. Using the phylogenetic analysis, we identified three classes of GATA factors in C. reinhardtii according to the structure of the zinc finger domain and showed their difference from the classification of GATA factors developed on vascular plants.

Extraction and selection of high-molecular-weight DNA for long-read sequencing from Chlamydomonas reinhardtii.

Recent advances in long-read sequencing technologies have enabled the complete assembly of eukaryotic genomes from telomere to telomere by allowing repeated regions to be fully sequenced and assembled, thus filling the gaps left by previous short-read sequencing methods. Furthermore, long-read sequencing can also help characterizing structural variants, with applications in the fields of genome evolution or cancer genomics. For many organisms, the main bottleneck to sequence long reads remains the lack of robust methods to obtain high-molecular-weight (HMW) DNA. For this purpose, we developed an optimized protocol to extract DNA suitable for long-read sequencing from the unicellular green alga Chlamydomonas reinhardtii, based on CTAB/phenol extraction followed by a size selection step for long DNA molecules. We provide validation results for the extraction protocol, as well as statistics obtained with Oxford Nanopore Technologies sequencing.

A cell-based model for size control in the multiple fission alga Chlamydomonas reinhardtii

Understanding how population-size homeostasis emerges from stochastic individual cell behaviors remains a challenge in biology.1,2,3,4,5,6,7 The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) proliferates using a multiple fission cell cycle, where a prolonged G1 phase is followed by n rounds of alternating division cycles (S/M) to produce 2n daughters. A "Commitment" sizer in mid-G1 phase ensures sufficient cell growth before completing the cell cycle. A mitotic sizer couples mother-cell size to division number (n) such that daughter size distributions are uniform regardless of mother size distributions. Although daughter size distributions were highly robust to altered growth conditions, ∼40% of daughter cells fell outside of the 2-fold range expected from a "perfect" multiple fission sizer.7,8 A simple intuitive power law model with stochastic noise failed to reproduce individual division behaviors of tracked single cells. Through additional iterative modeling, we identified an alternative modified threshold (MT) model, where cells need to cross a threshold greater than 2-fold their median starting size to become division-competent (i.e., Committed), after which their behaviors followed a power law model. The Commitment versus mitotic size threshold uncoupling in the MT model was likely a key pre-adaptation in the evolution of volvocine algal multicellularity. A similar experimental approach was used in size mutants mat3/rbr and dp1 that are, respectively, missing repressor or activator subunits of the retinoblastoma tumor suppressor complex (RBC). Both mutants showed altered relationships between Commitment and mitotic sizer, suggesting that RBC functions to decouple the two sizers.

Moderate high temperature is beneficial or detrimental depending on carbon availability in the green alga Chlamydomonas reinhardtii.

High temperatures impair plant growth and reduce agricultural yields, but the underlying mechanisms remain elusive. The unicellular green alga Chlamydomonas reinhardtii is an excellent model to study heat responses in photosynthetic cells due to its fast growth rate, many similarities in cellular processes to land plants, simple and sequenced genome, and ample genetic and genomics resources. Chlamydomonas grows in light by photosynthesis and with externally supplied acetate as an organic carbon source. Understanding how organic carbon sources affect heat responses is important for the algal industry but remains understudied. We cultivated wild-type Chlamydomonas under highly controlled conditions in photobioreactors at 25 °C (control), 35 °C (moderate high temperature), or 40 °C (acute high temperature) with or without constant acetate supply for 1 or 4 day. Treatment at 35 °C increased algal growth with constant acetate supply but reduced algal growth without sufficient acetate. The overlooked and dynamic effects of 35 °C could be explained by induced acetate uptake and metabolism. Heat treatment at 40 °C for more than 2 day was lethal to algal cultures with or without constant acetate supply. Our findings provide insights to understand algal heat responses and help improve thermotolerance in photosynthetic cells.

An RNA thermometer in the chloroplast genome of Chlamydomonas facilitates temperature-controlled gene expression.

Riboregulators such as riboswitches and RNA thermometers provide simple, protein-independent tools to control gene expression at the post-transcriptional level. In bacteria, RNA thermometers regulate protein synthesis in response to temperature shifts. Thermometers outside of the bacterial world are rare, and in organellar genomes, no RNA thermometers have been identified to date. Here we report the discovery of an RNA thermometer in a chloroplast gene of the unicellular green alga Chlamydomonas reinhardtii. The thermometer, residing in the 5' untranslated region of the psaA messenger RNA forms a hairpin-type secondary structure that masks the Shine-Dalgarno sequence at 25°C. At 40°C, melting of the secondary structure increases accessibility of the Shine-Dalgarno sequence to initiating ribosomes, thus enhancing protein synthesis. By targeted nucleotide substitutions and transfer of the thermometer into Escherichia coli, we show that the secondary structure is necessary and sufficient to confer the thermometer properties. We also demonstrate that the thermometer provides a valuable tool for inducible transgene expression from the Chlamydomonas plastid genome, in that a simple temperature shift of the algal culture can greatly increase recombinant protein yields.

Phosphoribulokinase abundance is not limiting the Calvin-Benson-Bassham cycle in Chlamydomonas reinhardtii.

Improving photosynthetic efficiency in plants and microalgae is of utmost importance to support the growing world population and to enable the bioproduction of energy and chemicals. Limitations in photosynthetic light conversion efficiency can be directly attributed to kinetic bottlenecks within the Calvin-Benson-Bassham cycle (CBBC) responsible for carbon fixation. A better understanding of these bottlenecks in vivo is crucial to overcome these limiting factors through bio-engineering. The present study is focused on the analysis of phosphoribulokinase (PRK) in the unicellular green alga Chlamydomonas reinhardtii. We have characterized a PRK knock-out mutant strain and showed that in the absence of PRK, Chlamydomonas cannot grow photoautotrophically while functional complementation with a synthetic construct allowed restoration of photoautotrophy. Nevertheless, using standard genetic elements, the expression of PRK was limited to 40% of the reference level in complemented strains and could not restore normal growth in photoautotrophic conditions suggesting that the CBBC is limited. We were subsequently able to overcome this initial limitation by improving the design of the transcriptional unit expressing PRK using diverse combinations of DNA parts including PRK endogenous promoter and introns. This enabled us to obtain strains with PRK levels comparable to the reference strain and even overexpressing strains. A collection of strains with PRK levels between 16% and 250% of WT PRK levels was generated and characterized. Immunoblot and growth assays revealed that a PRK content of ≈86% is sufficient to fully restore photoautotrophic growth. This result suggests that PRK is present in moderate excess in Chlamydomonas. Consistently, the overexpression of PRK did not increase photosynthetic growth indicating that that the endogenous level of PRK in Chlamydomonas is not limiting the Calvin-Benson-Bassham cycle under optimal conditions.

N-Acetyl-L-glutamate Kinase of Chlamydomonas reinhardtii: In Vivo Regulation by PII Protein and Beyond.

N-Acetyl-L-glutamate kinase (NAGK) catalyzes the rate-limiting step in the ornithine/arginine biosynthesis pathway in eukaryotic and bacterial oxygenic phototrophs. NAGK is the most highly conserved target of the PII signal transduction protein in Cyanobacteria and Archaeplastida (red algae and Chlorophyta). However, there is still much to be learned about how NAGK is regulated in vivo. The use of unicellular green alga Chlamydomonas reinhardtii as a model system has already been instrumental in identifying several key regulation mechanisms that control nitrogen (N) metabolism. With a combination of molecular-genetic and biochemical approaches, we show the existence of the complex CrNAGK control at the transcriptional level, which is dependent on N source and N availability. In growing cells, CrNAGK requires CrPII to properly sense the feedback inhibitor arginine. Moreover, we provide primary evidence that CrPII is only partly responsible for regulating CrNAGK activity to adapt to changing nutritional conditions. Collectively, our results suggest that in vivo CrNAGK is tuned at the transcriptional and post-translational levels, and CrPII and additional as yet unknown factor(s) are integral parts of this regulation.

Partial inhibition of the inter-photosystem electron transfer at cytochrome b6f complex promotes periodic surges of hydrogen evolution in Chlamydomonas reinhardtii

Periodic surges of H2 evolution were observed in the wild-type strain CC-5325 of the green unicellular alga Chlamydomonas reinhardtii in the presence of the electron transport inhibitors dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB, 3.5 μM) and 2,4-dinitrophenylether and iodonitrothymol (DNP-INT, 0.6 μM). Addition of DBMIB partly inhibited the electron transfer from Cytochrome b6f complex to Photosystem I, over-reduced the plastoquinone pool, gradually inhibited photosystem II and created anoxic conditions in cells. During 30 days of anaerobic incubation, continues H2 photoproduction with a minimum rate of 1 ml/L of culture per day was accompanied with additional outbursts of H2 evolution. The first noticeable peak of H2 evolution was observed on day 6 of incubation, with maximum rate of 23 ml of H2 per L of culture per day. It was repeated on day 9 and day 22 with the 2 and 4 times lower rates respectively. Addition of DNP-NT showed similar effect by inducing the H2 photoproduction for 15 days, albeit at much lower rates. Contribution of the direct and indirect pathways to the H2 production is shown by fluorescence, thermoluminescence and electron paramagnetic resonance spectroscopy. It is proposed that photosynthetic electron transport in combination with photorespiration, chlororespiration and starch accumulation can switch on and off between photosynthetic, H2 producing and survival modes of cell metabolism. Controlled switching between these modes could potentially maintain the long lasting photosynthetic H2 production in the wild-type of Chlamydomonas.

An All-Green Photo-Electrochemical Biosensor Using Microalgae Immobilized on Eco-Designed Lignin-Based Screen-Printed Electrodes to Detect Sustainable Nanoherbicides.

Herein, a novel completely green biosensor was designed exploiting both the biological and instrumental components made of eco-friendly materials for the detection of herbicides encapsulated into biodegradable nanoparticles for a sustainable agriculture. Similar nanocarriers, indeed, can deliver herbicides to the correct location, reducing the amount of active chemicals deposited in the plant, impacting the agricultural and food industries less. However, handling measurements of nanoherbicides is crucial to provide comprehensive information about their status in the agricultural fields to support farmers in decision-making. In detail, whole cells of the unicellular green photosynthetic alga Chlamydomonas reinhardtii UV180 mutant were immobilized by a green protocol on carbonized lignin screen-printed electrodes and integrated into a photo-electrochemical transductor for the detection of nanoformulated atrazine. Specifically, atrazine encapsulated into zein and chitosan doped poly-ε-caprolactone nanoparticles (atrazine-zein and atrazine-PCL-Ch) were analyzed following the current signals at a fixed applied potential of 0.8 V, in a range between 0.1 and 5 µM, indicating a linear relationship in the measured dose-response curves and a detection limit of 0.9 and 1.1 nM, respectively. Interference studies resulted in no interference from 10 ppb bisphenol A, 1 ppb paraoxon, 100 ppb arsenic, 20 ppb copper, 5 ppb cadmium, and 10 ppb lead at safety limits. Finally, no matrix effect was observed on the biosensor response from wastewater samples and satisfactory recovery values of 106 ± 8% and 93 ± 7% were obtained for atrazine-zein and atrazine-PCL-Ch, respectively. A working stability of 10 h was achieved.

Small RNA-mediated silencing of phototropin suppresses the induction of photoprotection in the green alga Chlamydomonas reinhardtii

Small RNAs (sRNAs) form complexes with Argonaute proteins and bind to transcripts with complementary sequences to repress gene expression. sRNA-mediated regulation is conserved in a diverse range of eukaryotes and is involved in the control of various physiological functions. sRNAs are present in the unicellular green alga Chlamydomonas reinhardtii, and genetic analyses revealed that the core sRNA biogenesis and action mechanisms are conserved with those of multicellular organisms. However, the roles of sRNAs in this organism remain largely unknown. Here, we report that Chlamydomonas sRNAs contribute to the induction of photoprotection. In this alga, photoprotection is mediated by LIGHT HARVESTING COMPLEX STRESS-RELATED 3 (LHCSR3), whose expression is induced by light signals through the blue-light receptor phototropin (PHOT). We demonstrate here that sRNA-defective mutants showed increased PHOT abundance leading to greater LHCSR3 expression. Disruption of the precursor for two sRNAs predicted to bind to the PHOT transcript also increased PHOT accumulation and LHCSR3 expression. The induction of LHCSR3 in the mutants was enhanced by light containing blue wavelengths, but not by red light, indicating that the sRNAs regulate the degree of photoprotection via regulation of PHOT expression. Our results suggest that sRNAs are involved not only in the regulation of photoprotection but also in biological phenomena regulated by PHOT signaling.

Spatial Distribution of Flagellated Microalgae Chlamydomonas reinhardtii in a Quasi-Two-Dimensional Space

Although the phenomenon of collective order formation by cell–cell interactions in motile cells, microswimmers, has been a topic of interest, most studies have been conducted under conditions of high cell density, where the space occupancy of a cell population relative to the space size ϕ > 0.1 (ϕ is the area fraction). We experimentally determined the spatial distribution (SD) of the flagellated unicellular green alga Chlamydomonas reinhardtii at a low cell density (ϕ ≈ 0.01) in a quasi-two-dimensional (thickness equal to cell diameter) restricted space and used the variance-to-mean ratio to investigate the deviation from the random distribution of cells, that is, do cells tend to cluster together or avoid each other? The experimental SD is consistent with that obtained by Monte Carlo simulation, in which only the excluded volume effect (EV effect) due to the finite size of cells is taken into account, indicating that there is no interaction between cells other than the EV effect at a low cell density of ϕ ≈ 0.01. A simple method for fabricating a quasi-two-dimensional space using shim rings was also proposed.

Evaluation of Shifts of Gene Transcription Levels of Unicellular Green Alga Chlamydomonas reinhardtii Due to UV-C Irradiation

Green algae produce valuable lipids as carbon-recycling resources. Collecting whole cells with the intracellular lipids could be efficient without cell burst; however, direct use of the cells causes microbial contamination in environments. Then, UV-C irradiation was selected to satisfy the requirements of avoiding the cell burst and sterilizing cells with Chlamydomonas reinhardtii. UV-C irradiation with 1.209 mW·cm-2 showed enough sterilization activity for 1.6 × 107 cells·mL-1 of C. reinhardtii in a depth of 5 mm for 10 min. The irradiation showed no effects to composition and contents of the intracellular lipids. From the viewpoint of transcriptomic analysis, the irradiation displayed possibilities of (i) inhibition of the synthesis of lipids due to decrement of the transcription of related genes, such as diacylglycerol acyl transferase and cyclopropane fatty acid synthase, and (ii) activation of lipid degradation and the production of NADH2+ and FADH2 due to increment of the transcription of related genes, such as isocitrate dehydrogenase, dihydrolipoamide dehydrogenase and malate dehydrogenase. Irradiation until cell death could be insufficient to shift the metabolic flows even though the transcriptions were already shifted to lipid degradation and energy production. This paper is the first report of the response of C. reinhardtii to UV-C irradiation on the transcription level.

Microalgal glycerol-3-phosphate acyltransferase role in galactolipids and high-value storage lipid biosynthesis.

Glycerolipids are the most abundant lipids in microalgae, and glycerol-3-phosphate:acyl-CoA acyltransferase (GPAT) plays an important role in their biosynthesis. However, the biochemical and biological functions of algal GPAT remain poorly characterized. Here, we characterized the endoplasmic reticulum (ER)-associated GPAT of the model unicellular green alga Chlamydomonas reinhardtii (CrGPATer). Enzymatic assays indicated that CrGPATer is an sn-1 acyltransferase using a variety of acyl-CoAs as the acyl donor. Subcellular localization revealed that CrGPATer was associated with ER membranes and lipid droplets. We constructed overexpression (OE) and knockdown (KD) transgenic C. reinhardtii lines to investigate the in vivo function of CrGPATer. Lipidomic analysis indicated that CrGPATer OE enhanced the cellular content of galactolipids, especially monogalactosyldiacylglycerol, under nitrogen deficiency stress. Correspondingly, CrGPATer KD lines contained lower contents of galactolipids than the control. Feeding experiments with labeled phosphatidic acid revealed that the intermediate of the eukaryotic Kennedy pathway could be used for galactolipid biosynthesis in the chloroplasts. These results provided multiple lines of evidence that the eukaryotic Kennedy pathway mediated by CrGPATer may be involved in galactolipid biosynthesis in C. reinhardtii. OE of CrGPATer significantly increased the content of triacylglycerol and the yield of biomass. Moreover, the content and yield of 1, 3-olein-2-palmitin, a high-value lipid that can be used as an alternative for human milk fat in infant formula, were significantly enhanced in the OE transgenic lines. Taken together, this study provided insights into the biochemical and biological functions of CrGPATer and its potential as a genetic engineering target in functional lipid manufacturing.

Two specific domains of the γ subunit of chloroplast FoF1 provide redox regulation of the ATP synthesis through conformational changes

Chloroplast FoF1-ATP synthase (CFoCF1) converts proton motive force into chemical energy during photosynthesis. Although many studies have been done to elucidate the catalytic reaction and its regulatory mechanisms, biochemical analyses using the CFoCF1 complex have been limited because of various technical barriers, such as the difficulty in generating mutants and a low purification efficiency from spinach chloroplasts. By taking advantage of the powerful genetics available in the unicellular green alga Chlamydomonas reinhardtii, we analyzed the ATP synthesis reaction and its regulation in CFoCF1. The domains in the γ subunit involved in the redox regulation of CFoCF1 were mutated based on the reported structure. An invivo analysis of strains harboring these mutations revealed the structural determinants of the redox response during the light/dark transitions. In addition, we established a half day purification method for the entire CFoCF1 complex from C. reinhardtii and subsequently examined ATP synthesis activity by the acid-base transition method. We found that truncation of the β-hairpin domain resulted in a loss of redox regulation of ATP synthesis (i.e., constitutively active state) despite retaining redox-sensitive Cys residues. In contrast, truncation of the redox loop domain containing the Cys residues resulted in a marked decrease in the activity. Based on this mutation analysis, we propose a model of redox regulation of the ATP synthesis reaction by the cooperative function of the β-hairpin and the redox loop domains specific to CFoCF1.

Chlamydomonas reinhardtii mutants deficient for Old Yellow Enzyme 3 exhibit increased photooxidative stress.

Old Yellow Enzymes (OYEs) are flavin-containing ene-reductases that have been intensely studied with regard to their biotechnological potential for sustainable chemical syntheses. OYE-encoding genes are found throughout the domains of life, but their physiological role is mostly unknown, one reason for this being the promiscuity of most ene-reductases studied to date. The unicellular green alga Chlamydomonas reinhardtii possesses four genes coding for OYEs, three of which we have analyzed biochemically before. Ene-reductase CrOYE3 stood out in that it showed an unusually narrow substrate scope and converted N-methylmaleimide (NMI) with high rates. This was recapitulated in a C. reinhardtii croye3 mutant that, in contrast to the wild type, hardly degraded externally added NMI. Here we show that CrOYE3-mediated NMI conversion depends on electrons generated photosynthetically by photosystem II (PSII) and that the croye3 mutant exhibits slightly decreased photochemical quenching in high light. Non-photochemical quenching is strongly impaired in this mutant, and it shows enhanced oxidative stress. The phenotypes of the mutant suggest that C. reinhardtii CrOYE3 is involved in the protection against photooxidative stress, possibly by converting reactive carbonyl species derived from lipid peroxides or maleimides from tetrapyrrole degradation.

Chloroplast genomes and GMOs. History, features and perspectives on plastid transgenesis in plant biotechnology

Chloroplasts provide life on our planet, by creating carbohydrates, amino acids, lipids and O2 through the process of photosynthesis. Two billion years ago, a eukaryotic ancestor entered symbiosis with photosynthetic cyanobacterium, giving rise to chloroplasts. During evolution, chloroplasts transferred most protein-coding genes of free-living bacteria to the nucleus, but retained a semi-autonomous genetic status their own genomes and transcription and translation machinery.
 The genetics of plastids began in 1909, when Baur and Correns described the non-Mendelian, maternal inheritance of Pelargonium leaf color: green, white and variegated. The presence DNA in chloroplasts (chDNA) was prove in the 1960s, and later have been shown, that male chDNA in zygotes is specifically degraded, assuring maternal inheritance in most plants.
 The plastid transformation was first described in the unicellular green alga Chlamydomonas reinhardtii [1]. This method of plant biotechnology is an alternative to traditional introduction of foreign DNA into the nucleus, and has a number of advantages: (a) the large copy number of chDNA offers high-level transgene expression; (b) site-specific integration of foreign genes reduces the number of transgenic lines to be evaluated; (c) the lack of gene silencing or position effects in chloroplast genomes; (d) the chloroplast employs a prokaryotic gene expression system, and allows the expression of polycistronic genes [2]. Thus, chloroplasts are becoming attractive hosts for the introduction of new agronomic traits, as well as for the biosynthesis of high-value pharmaceuticals, biomaterials and industrial enzymes.

Gating and ion selectivity of Channelrhodopsins are critical for photo-activated orientation of Chlamydomonas as shown by in vivo point mutation

The green unicellular alga Chlamydomonas reinhardtii with two photoreceptors called channelrhodopsins is a model organism that gave birth to a new scientific field of biomedical studies, optogenetics. Although channelrhodopsins are helping to decipher the activity of the human brain, their functionality has never been extensively studied in the organism of origin, mainly due to the difficulties connected to reverse genetic interventions. In this study, we present a CRISPR-Cas9-based technique that enables a precise in vivo exchange of single amino acids in a selected gene. To shed light on the function of channelrhodopsins ChR1 (C1) and ChR2 (C2) in vivo, we deleted both channelrhodopsins independently in the wild-type strain and introduced point mutations in the remaining channel, causing modified photocycle kinetics and ion selectivity. The mutated strains, ΔC1C2-E123T, ΔC1C2-E90R and ΔC1C2-E90Q, showed about 100-fold decrease in photosensitivity, a reduced photophobic response and faster light adaptation rates due to accelerated photocycle kinetics and reduced Ca2+ conductance. Moreover, the ΔC1C2-E90Q with an additionally reduced H+ permeability produced an electrical response only in the presence of Na+ ions, highlighting a contribution and importance of H+ conductance to photocurrents in the wild-type algae. Finally, in the ΔC1C2-E90R strain with the channelrhodopsin selectivity converted to anions, no photo-responses were detected. We conclude that the precise photocycle kinetics and the particular ion selectivity of channelrhodopsins are the key parameters for efficient phototaxis in low light conditions.

A gap-free genome assembly of Chlamydomonas reinhardtii and detection of translocations induced by CRISPR-mediated mutagenesis

Genomic assemblies of the unicellular green alga Chlamydomonas reinhardtii have provided important resources for researchers. However, assembly errors, large gaps, and unplaced scaffolds as well as strain-specific variants currently impede many types of analysis. By combining PacBio HiFi and Oxford Nanopore long-read technologies, we generated a de novo genome assembly for strain CC-5816, derived from crosses of strains CC-125 and CC-124. Multiple methods of evaluating genome completeness and base-pair error rate suggest that the final telomere-to-telomere assembly is highly accurate. The CC-5816 assembly enabled previously difficult analyses that include characterization of the 17 centromeres, rDNA arrays on three chromosomes, and 56 insertions of organellar DNA into the nuclear genome. Using Nanopore sequencing, we identified sites of cytosine (CpG) methylation, which are enriched at centromeres. We analyzed CRISPR-Cas9 insertional mutants in the PF23 gene. Two of the three alleles produced progeny that displayed patterns of meiotic inviability that suggested the presence of a chromosomal aberration. Mapping Nanopore reads from pf23-2 and pf23-3 onto the CC-5816 genome showed that these two strains each carry a translocation that was initiated at the PF23 gene locus on chromosome 11 and joined with chromosomes 5 or 3, respectively. The translocations were verified by demonstrating linkage between loci on the two translocated chromosomes in meiotic progeny. The three pf23 alleles display the expected short-cilia phenotype, and immunoblotting showed that pf23-2 lacks the PF23 protein. Our CC-5816 genome assembly will undoubtedly provide an important tool for the Chlamydomonas research community.

Development of a Simple Fabrication Method for Magnetic Micro Stir Bars and Induction of Rotational Motion in Chlamydomonas reinhardtii.

A magnetic micro stirrer bar (MMSB) is used in the mixing operation of microfluidic devices. We have established a low-cost and easy method to make MMSBs using magnetic (neodymium magnets, magnet sheets) or non-magnetic powders (SUS304) as materials. We demonstrated three kinds of MMSB have respective advantages. To confirm the practical use of this MMSB, a cell suspension of the motile unicellular green alga Chlamydomonas reinhardtii was stirred in microwells. As a result, the number of rotating cells increased with only one of the two flagella mechanically removed by the shear force of the rotating bar, which facilitates the kinetic analysis of the flagellar motion of the cell. The rotational motion of the monoflagellate cell was modeled as translational (orbital) + spinning motion of a sphere in a viscous fluid and the driving force per flagellum was confirmed to be consistent with previous literature. Since the present method does not use genetic manipulations or chemicals to remove a flagellum, it is possible to obtain cells in a more naturally viable state quickly and easily than before. However, since the components eluted from the powder material harm the health of cells, it was suggested that MMSB coated with resin for long-term use would be suitable for more diverse applications.