Unicellular Alga Research Articles

Effects of food level and fecal material on the plasticity of metamorphic traits in Asiatic toad, Bufo gargarizans, in Central China

Abstract According to Richards’ hypothesis, the presence of a nonpigmented unicellular alga in the intestinal tract of tadpoles is believed to be the inhibitory factor that affects their growth. It is proposed that thecanimals eat their fecal material containing these algal cells, which are then reintroduced into their systems when the alga is shed with the feces and dropped to the bottom. In this study, we aimed to investigate the plasticity of metamorphic traits in the Asiatic toad (Bufo gargarizans) under different combinations of fecal material and food levels. Our findings revealed that B. gargarizans tadpoles reared at high food levels exhibited accelerated larval periods but had smaller metamorphic sizes compared to those reared at low food levels. This suggests that the tadpoles prioritize rapid development at the expense of body mass during metamorphosis. Furthermore, our study discovered evidence supporting the positive effect of fecal material on tadpole growth, as indicated by the investigation of growth rate. Importantly, the presence of feces did not result in smaller body mass on average for Asiatic toad tadpoles compared to those raised in the absence of fecal material. The difference in body mass between fecal material treatments tended to be smaller at low food levels, implying that feces does not inhibit growth in B. gargarizans tadpoles. Therefore, our results contradict Richards’ hypothesis and provide support for a novel mechanism that is selected for in areas where coprophagy is likely to be advantageous, particularly in times of food scarcity.

Cytochromes b5 Occurrence in Viruses Belonging to the Order Megavirales.

Cytochrome b5 is a small electron transport protein that is found in animals, plants, fungi and photosynthetic proteobacteria where it plays key metabolic roles in energy production, lipid and sterol biosynthesis and cytochrome P450 biochemistry. Previously it was shown that a gene encoding a soluble and functional cytochrome b5 protein was encoded in the large double stranded DNA virus OtV2 that infects the unicellular marine green alga Ostreococcus tauri, the smallest free-living eukaryote described to-date. This single gene represented a unique finding in the virosphere. We now report that genes for soluble and membrane-bound cytochromes b5 also occur in giant viruses in the proposed order Megavirales, particularly the AT-rich Mimiviridae and Tupanviruses. Conversely, other members of the Megaviralestaxa such as the GC-rich Pandoraviridae have not been found to encode cytochrome b5 as yet. Megaviruses encoding cytochrome b5 have been isolated from the deep ocean, from freshwater and terrestrial sources, as well as from human patients. Giant virus cytochrome b5 proteins share high sequence identity with one another (45-95% depending on group) but no more than 25% identity with the cytochrome b5 gene product we identified in Acanthamoeba castellanii, an amoeba host for many giant viruses. Thus, the origin of the unique cytochrome b5 genes in giant viruses remainsunknown. Examination of viral cytochrome b5 primary amino acid sequences revealed that some have either a N- or C-terminal transmembrane anchor, whilst others lack a membrane anchor and are thus predicted to be soluble proteins. This cytochrome b5 topography suggests adapted biochemical functions in those viruses. Our findings raise questions regarding the evolution and diversity of cytochrome b5 proteins in nature, adding to questions about the origin of viral haemoproteins in general.

KiNext: a portable and scalable workflow for the identification and classification of protein kinases

BackgroundProtein kinases are a diverse superfamily of proteins common to organisms across the tree of life that are typically involved in signal transduction, allowing organisms to sense and respond to biotic or abiotic environmental factors. They have important roles in organismal physiology, including development, reproduction, acclimation to environmental stress, while their dysregulation can lead to disease, including several forms of cancer. Identifying the complement of protein kinases (the kinome) of any organism is useful for understanding its physiological capabilities, limitations and adaptations to environmental stress. The increasing availability of genomes makes it now possible to examine and compare the kinomes across a broad diversity of organisms. Here we present a pipeline respecting the FAIR principles (findable, accessible, interoperable and reusable) that facilitates the search and identification of protein kinases from a predicted proteome, and classifies them according to group of serine/threonine/tyrosine protein kinases present in eukaryotes.ResultsKiNext is a Nextflow pipeline that regroups a number of existing bioinformatic tools to search for and classify the protein kinases of an organism in a reproducible manner, starting from a set of amino acid sequences. Conventional eukaryotic protein kinases (ePKs) and atypical protein kinases (aPKs) are identified by using Hidden Markov Models (HMMs) generated from the catalytic domains of kinases. Furthermore, KiNext categorizes ePKs into the eight kinase groups by employing dedicated Hidden Markov Models (HMMs) tailored for each group. The performance of the KiNext pipeline was validated against previously identified kinomes obtained with other tools that were already published for two marine species, the Pacific oyster Crassostrea gigas and the unicellular green alga Ostreoccocus tauri. KiNext outperformed previous results by finding previously unidentified kinases and by attributing a large proportion of previously unclassified kinases to a group in both species. These results demonstrate improvements in kinase identification and classification, all while providing traceability and reproducibility of results in a FAIR pipeline. The default HMM models provided with KiNext are most suitable for eukaryotes, but the pipeline can be easily modified to include HMM models for other taxa of interest.ConclusionThe KiNext pipeline enables efficient and reproducible identification of kinomes based on predicted amino acid sequences (i.e. proteomes). KiNext was designed to be easy to use, automated, portable and scalable.

Forensic Diatom Analysis: Where Do We Stand and What Are the Latest Diagnostic Advances?

Background: diatoms are unicellular algae that have been used for more than a century for forensic purposes to diagnose drowning, with more or less success depending on the historical era. Although many years have passed, scientific research on diatoms has never ceased, which testifies to their enduring allure in forensics. Of course, diatom research has evolved and expanded over time, changing with the availability of new techniques and technologies. The volume of articles and their production over a period of many years has resulted in old, current, and new knowledge on diatoms being scattered over a large number of books and articles. Objectives: the purpose of this narrative literature review is, therefore, to summarize all this information and bring it together in a single work that can be useful for those who are studying diatoms and their usefulness for forensics for the first time, for those who are looking for proven methods of analysis, and finally for those who are interested in exploring new frontiers of research. Methods: a comprehensive literature search that included all studies dealing with the applications of diatoms in forensic science was performed in the most popular electronic databases. Results: traditional methods have been complemented by molecular and imaging methods and, more recently, by artificial intelligence. In addition, new biological substrates have been found for the analysis of diatoms. Conclusions: all this has led, on the one hand, to the consolidation of a whole body of knowledge on diatoms, on which this forensic analysis is still based, and, on the other hand, has opened up numerous new research directions.

Structure of a step II catalytically activated spliceosome from Chlamydomonas reinhardtii.

Pre-mRNA splicing, a fundamental step in eukaryotic gene expression, is executed by the spliceosomes. While there is extensive knowledge of the composition and structure of spliceosomes in yeasts and humans, the structural diversity of spliceosomes in non-canonical organisms remains unclear. Here, we present a cryo-EM structure of a step II catalytically activated spliceosome (C* complex) derived from the unicellular green alga Chlamydomonas reinhardtii at 2.6 Å resolution. This Chlamydomonas C* complex comprises 29 proteins and four RNA elements, creating a dynamic assembly that shares a similar overall architecture with yeast and human counterparts but also has unique features of its own. Distinctive structural characteristics include variations in protein compositions as well as some noteworthy RNA features. The splicing factor Prp17, with four fragments and a WD40 domain, is engaged in intricate interactions with multiple protein and RNA components. The structural elucidation of Chlamydomonas C* complex provides insights into the molecular mechanism of RNA splicing in plants and understanding splicing evolution in eukaryotes.

The ascorbate peroxidase-related protein: insights into its functioning in Chlamydomonas and Arabidopsis.

We review the newly classified ascorbate peroxidase-related (APX-R) proteins, which do not use ascorbate as electron donor to scavenge H2O2. We summarize recent discoveries on the function and the characterization of the APX-R protein of the green unicellular alga Chlamydomonas reinhardtii and the land plant Arabidopsis thaliana. Additionally, we conduct in silico analyses on the conserved MxxM motif, present in most of the APX-R protein in different organisms, which is proposed to bind copper. Based on these analyses, we discuss the similarities between the APX-R and the class III peroxidases.

Genomic and biogeographic characterisation of the novel prasinovirus Mantoniella tinhauana virus 1.

Mamiellophyceae are a ubiquitous class of unicellular green algae in the global ocean. Their ecological importance is highlighted in studies focused on the prominent genera Micromonas, Ostreococcus, and Bathycoccus. Mamiellophyceae are susceptible to prasinoviruses, double-stranded DNA viruses belonging to the nucleocytoplasmic large DNA virus group. Our study represents the first isolation of a prasinovirus in the South China Sea and the only one to infect the globally distributed genus Mantoniella. We conducted a comparative analysis with previously identified viral relatives, encompassing morphological characteristics, host specificity, marker-based phylogenetic placement, and whole-genome sequence comparisons. Although it shares morphological and genetic similarities with established prasinoviruses, this novel virus showed distinct genetic traits, confining its infection to the species Mantoniella tinhauana. We also explored the global biogeography of this prasinovirus and its host by mapping metagenomic data and analysing their relationship with various environmental parameters. Our results demonstrate a pronounced link between the virus and its host, both found predominantly in higher latitudes in the surface ocean. By gaining an increased understanding of the relationships between viruses, hosts, and environments, researchers can better make predictions and potentially implement measures to mitigate the consequences of climate change on oceanic processes.

Spirulina protein concentrate hydrogel reinforced by hydroxyapatite nanoparticles and hydroxychloroquin: A novel platform for bone regeneration

Among the fabrication methods, hydrogels have received significant attention due to their excellent ability to mimic the extracellular matrix structure. Spirulina is a type of unicellular blue-green algae. It is an excellent source of protein and a wide variety of other nutrients. Hydroxychloroquin (HCQ) reduces bone resorption and osteoclasts growth. This study hypothesized that the hydrogel, which is composed of Spirulina protein cross-linked by transglutaminase and reinforced by hydroxyapatite (HAP) nanoparticles loaded with HCQ, could potentially aid in bone regeneration. The protein from Spirulina was extracted using the probe sonication method. Afterwards, hydrogels were prepared by incorporating the Spirulina protein with gelatin and cross-linked with varying percentages of transglutaminase. HCQ-loaded HAP nanoparticles were used to enhance not only the mechanical properties of the hydrogels but also to stimulate bone mineralization. Hydrogels were studied for their mechanical and rheological properties in vitro, as well as their ability to swell, break down, injectability, and release HCQ. Cell adhesion, proliferation, and bone mineralization on MG-63 osteoblast cells were also studied using Alizarine red and alkaline phosphatase markers. The loading efficiency of HCQ in HAP nanoparticles was about 97 %. The HCQ release rate from hydrogel containing HAP was 85 % in 9 hours. The tensile strength and injectability force of the optimized hydrogel were in the range of 530.75 KPa and 21 N, respectively. The results showed that hydrogels made with 10 % protein, 150 units of transglutaminase, 6 % gelatin, and HAP loaded with HCQ had the best cell behavior and improved markers of bone regeneration. The promising results obtained suggest that the prepared hydrogel may act as a suitable construct for accelerating bone regeneration.

Genome of Halimeda opuntia reveals differentiation of subgenomes and molecular bases of multinucleation and calcification in algae

Algae mostly occur either as unicellular (microalgae) or multicellular (macroalgae) species, both being uninucleate. There are important exceptions, however, as some unicellular algae are multinucleate and macroscopic, some of which inhabit tropical seas and contribute to biocalcification and coral reef robustness. The evolutionary mechanisms and ecological significance of multinucleation and associated traits (e.g., rapid wound healing) are poorly understood. Here, we report the genome of Halimeda opuntia, a giant multinucleate unicellular chlorophyte characterized by interutricular calcification. We achieve a high-quality genome assembly that shows segregation into four subgenomes, with evidence for polyploidization concomitant with historical sea level and climate changes. We further find myosin VIII missing in H. opuntia and three other unicellular multinucleate chlorophytes, suggesting a potential mechanism that may underpin multinucleation. Genome analysis provides clues about how the unicellular alga could survive fragmentation and regenerate, as well as potential signatures for extracellular calcification and the coupling of calcification with photosynthesis. In addition, proteomic alkalinity shifts were found to potentially confer plasticity of H. opuntia to ocean acidification (OA). Our study provides crucial genetic information necessary for understanding multinucleation, cell regeneration, plasticity to OA, and different modes of calcification in algae and other organisms, which has important implications in reef conservation and bioengineering.

Euglena gracilis Enhances Innate and Adaptive Immunity through Specific Expression of Dectin-1 in CP-Induced Immunosuppressed Mice.

Euglena gracilis (E. gracilis), a species of unicellular algae, can accumulate large amounts of β-1,3-glucan paramylon, a polysaccharide, in its cytoplasm and has recently attracted interest as a bioproduct due to its various health benefits. In this study, the immune-enhancing effect of E. gracilis powder (EP) was investigated in vitro and in vivo. In vitro, the production of NO and cytokines and the mechanism of the signaling pathway of β-1,3-glucan were identified in RAW264.7 cells. In vivo, cyclophosphamide-induced (CP-induced) immunosuppressed C57BL/6 female mice were orally administered with three different concentrations (100, 300, and 600 mg/kg) of EP daily. After 14 days, the organs and whole blood were collected from each animal for further study. The weight loss of CP-treated mice was reversed by treatment with EP to levels comparable to those of control mice. In addition, the frequencies of NK1.1+, CD3+, CD4+, CD8+, and B220+ in immune cells isolated from the spleen were increased by EP treatment compared with water or RG. The secretion of TNF-α, IFN-γ, and IL-12 from splenocytes was also increased by EP treatment, as was the level of IgM in the serum of the mice. Finally, EP treatment specifically upregulated the expression of dectin-1 in the liver of CP-treated mice. E. gracilis could be a good candidate for a natural immune stimulator in the innate and adaptive response by secreting TNF-α, IFN-γ, and IL-12 through stimulating dectin-1 expression on the surface of immune cells.

Intracellular morphogenesis of diatom silica is guided by local variations in membrane curvature

Silica cell-wall formation in diatoms is a showcase for the ability of organisms to control inorganic mineralization. The process of silicification by these unicellular algae is tightly regulated within a membrane-bound organelle, the silica deposition vesicle (SDV). Two opposing scenarios were proposed to explain the tight regulation of this intracellular process: a template-mediated process that relies on preformed scaffolds, or a template-independent self-assembly process. The present work points to a third scenario, where the SDV membrane is a dynamic mold that shapes the forming silica. We use in-cell cryo-electron tomography to visualize the silicification process in situ, in its native-state, and with a nanometer-scale resolution. This reveals that the plasma membrane interacts with the SDV membrane via physical tethering at membrane contact sites, where the curvature of the tethered side of the SDV membrane mirrors the intricate silica topography. We propose that silica growth and morphogenesis result from the biophysical properties of the SDV and plasma membranes.

Natural organic matter (NOM) can increase the uptake fluxes of three critical metals (Ga, La, Pt) in a unicellular green alga

Critical metals such as gallium, lanthanum and platinum are considered essential in a modern economy and for the required energy transition. Their relatively recent and increasing use in new technologies have led to an increase in their environmental mobility. As they reach aquatic systems, these metals can interact with organic ligands and especially Natural Organic Matter (NOM). The formation of organic complexes would be expected to reduce metal bioavailability and uptake by living cells, according to the Biotic Ligand Model (BLM). However, exceptions to this model have been determined for several critical metals in the past. The present work compared internalization kinetics of Ga, La and Pt in the green alga Chlamydomonas reinhardtii in the presence of NOMs from different origins: humic and fulvic acids from Suwannee River as well as NOMs from Ontario (Bannister Lake and Luther Marsh). Complexation was determined using a partial ultrafiltration method allowing for a normalization of data based on speciation to compare all conditions based on the concentration of the metal that was not bound to NOM. While internalization metal fluxes varied greatly from one NOM source to the other, uptake was almost always significantly higher than expected based on metal speciation. Quite often, metal internalization fluxes were even significantly increased in the presence of NOM, for the same total metal exposure concentration. For instance, Pt internalization was twice greater in the presence of Bannister Lake NOM than it was in the absence of NOM. The assumption that such exceptions could be explained by NOM characteristics was contradicted by the variable results from one metal to another. To further explore this phenomenon, internalization mechanisms for these individual metals need to be elucidated. This is a necessary step to accurately estimate the risk posed by the presence of these metals in humic aquatic systems.

Late Pleistocene–Holocene palynology and paleoceanography of İmralı Basin, Marmara Sea: Pollen-spore, dinoflagellate cyst and other NPP zonations

The Marmara Sea is the oceanographic gateway that links the hypersaline Eastern Mediterranean Sea with the hyposaline Black Sea via Straits of Dardanelles and Bosphorus, respectively, and also occupies a transitional vegetation and climatic regime between the Mediterranean and Euxinic phytogeographic ecosystems. Recent palynological studies of long cores from the shallow İmralı Basin perched on the southeastern slope of the Marmara Sea reveal that major paleoecological changes accompanied the isolation and reconnection of the Marmara Sea during the last two Pleistocene glacial–interglacial cycles. Here, we use data primarily from two İmralı Basin cores to determine new quantitative zonations for the pollen-spore, dinoflagellate cyst (dinocyst) and other non-pollen palynomorph (NPP) assemblages in sediments representing the last ∼30 cal ka. A gap in one core is closed using data from three short gravity cores. Twenty-four radiocarbon ages and 20 correlation-ties for sapropel and ash layers from these five cores provide an essentially complete record for the past 30 cal ka. Use of a full suite of palynomorph data (pollen and spores, fungal spores, dinocysts and other phytoplankton, aquatic parasites/saprophytes, zoo-planktonic and zoo-benthic NPPs) shows for the first time that fluctuations in aquatic palynoassemblages during the glacial periods are more complex than simple salinity or temperature responses. The changes involve food web-level turnovers in trait- and size-based food web components in which potential parasites/saprophytes appear critically important.Pollen stratigraphies reveal a significant loss of Euxinic tree taxa by MIS 3c, with only partial recovery in MIS 1. The Euxinic forest elements are replaced by cultivars and other indicators of anthropogenic influences during the MIS 1 that is also marked by a dry interval and corresponding in part with the 4.2 cal ka Eastern Mediterranean Drought Event. The dinocyst zones for the last ∼30 cal ka show a clear turnover from assemblages dominated by brackish Ponto–Caspian taxa to marine assemblages at ∼11.7 cal ka. This phytoplanktonic signal lags slightly behind (a) the vegetation and shift in the other NPP commencing ∼13.2 cal ka, the initial marine incursion at 13.8 cal ka, and the arrival of coccolithophores at ∼13 cal ka. Comparison to published MIS 6 and 5 data reveals higher MIS 2–1 species diversity, reappearance of the MIS 6 marker Impagidinium inaequalis, and the emergences of Pyxidinopsis psilata f. quadrata, cf. Boreadinium breve, and Peridinium spp. of Brenner 2005. The pre-reconnection microbiota is dominated by the unicellular alga Sigmopollis, spikes of colonial Pediastrum algae, the saprophytes/parasites cf. Multiplicisphaeridium and Chytridiomycota. This microbiota is displaced by the post-reconnection assemblage dominated by Botryococcus coenobia, various Platyhelminth egg capsules and ostracod lining-types that persist through the Holocene. A distinctive transitional microbiota with Discocotyle-type turbellarian egg capsules marks the Bølling–Allerød warming, followed at ∼13.3 ka by a marine assemblage with micro-foraminiferal linings and crustacean eggs. The interglacial marine phase of MIS 1 in İmralı Basin has much higher dinocyst species diversity than MIS 5e, reflecting Neolithic impacts (agriculture, land clearance) and expanded shipping trade. Initial marine flooding in the Marmara Sea during both interglacials are marked by sapropel deposition but variations of ravinement surface erosion, and marine deep water residence time in a stratified Marmara Sea are potentially important factors underlying hypoxia/anoxia and sapropel deposition.

Mixed microalgal culture enhances biofilm adhesion during treatment of livestock wastewater: The role of extracellular polymeric substances

Microalgal biofilm can remove nutrients and refractory pollutants from livestock wastewater, transforming them into biomass or fuel. However, these applications are hampered by biofilm shedding. Coculture enhances effects, and its influence on the adhesion of attached microalgal biofilms requires exploration. In this study, two unicellular algae, Euglena (E) and Chlorella pyrenoidosa (C). and two filamentous algae, Klebsodium sp. (K) and Phormidium sp. (P), were paired two by two, aiming to investigate the effect of mixing on the adhesion of microalgal biofilm. The results showed that suitable microalgal combinations significantly enhanced the performance of the co-cultured microalgal biofilm. Biomass enhancement of 35 % and 21.9 % was observed in the PC group compared to the individual P and C groups, respectively. The content of tightly bound extracellular polymeric substances (TB-EPS) and its protein and polysaccharide content significantly correlating with the net biomass increase in all treatments, p < 0.05. Furthermore, measurement of zeta potentials and water contact angles indicated that alterations in protein secondary structures contributed to a lower intermolecular repulsion and greater hydrophobicity of the TB-EPS, which together with the stability provided by the higher β-glucan content supported the adhesion of the co-cultured microalgal biofilm.

Latent infection of an active giant endogenous virus in a unicellular green alga.

Latency is a common strategy in a wide range of viral lineages, but its prevalence in giant viruses remains unknown. Here we describe the activity and viral production from a 617 kbp integrated giant viral element in the model green alga Chlamydomonas reinhardtii. We resolve the integrated viral region using long-read sequencing and show that viral particles are produced and released in otherwise healthy cultures. A diverse array of viral-encoded selfish genetic elements are expressed during GEVE reactivation and produce proteins that are packaged in virions. In addition, we show that field isolates of Chlamydomonas sp. harbor latent giant viruses related to the C. reinhardtii GEVE that exhibit similar infection dynamics, demonstrating that giant virus latency is prevalent in natural host communities. Our work reports the largest temperate virus documented to date and the first active GEVE identified in a unicellular eukaryote, substantially expanding the known limits of viral latency.

π-π Interactions Drive the Homotypic Phase Separation of the Prion-like Diatom Pyrenoid Scaffold PYCO1

CO2 fixation in most unicellular algae relies on the pyrenoid, a biomolecular condensate, which sequesters the cell’s carboxylase Rubisco. In the marine diatom Phaeodactylum tricornutum, the pyrenoid tandem repeat protein Pyrenoid Component 1 (PYCO1) multivalently binds Rubisco to form a heterotypic Rubisco condensate. PYCO1 contains prion-like domains and can phase-separate homotypically in a salt-dependent manner. Here we dissect PYCO1 homotypic liquid–liquid phase separation (LLPS) by evaluating protein fragments and the effect of site-directed mutagenesis. Two of PYCO1′s six repeats are required for homotypic LLPS. Mutagenesis of a minimal phase-separating fragment reveals tremendous sensitivity to the substitution of aromatic residues. Removing positively charged lysines and arginines instead enhances the propensity of the fragment to condense. We conclude that PYCO1 homotypic LLPS is mostly driven by π-π interactions mediated by tyrosine and tryptophan stickers. In contrast π-cation interactions involving arginine or lysine are not significant drivers of LLPS in this system.

Establishment of an RNA-based transient expression system in the green alga Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, a unicellular green alga, is a prominent model for green biotechnology and for studying organelles’ function and biogenesis, such as chloroplasts and cilia. However, the stable expression of foreign genes from the nuclear genome in C. reinhardtii faces several limitations, including low expression levels and significant differences between clones due to genome position effects, epigenetic silencing, and time-consuming procedures. We developed a robust transient expression system in C. reinhardtii to overcome these limitations. We demonstrated efficient entry of in vitro-transcribed mRNA into wall-less cells and enzymatically dewalled wild-type cells via electroporation. The endogenous or exogenous elements can facilitate efficient transient expression of mRNA in C. reinhardtii, including the 5’ UTR of PsaD and the well-characterized Kozak sequence derived from the Chromochloris zofingiensis. In the optimized system, mRNA expression was detectable in 120 h with a peak around 4 h after transformation. Fluorescently tagged proteins were successfully transiently expressed, enabling organelle labeling and real-time determination of protein sub-cellular localization. Remarkably, transiently expressed IFT46 compensated for the ift46–1 mutant phenotype, indicating the correct protein folding and function of IFT46 within the cells. Additionally, we demonstrated the feasibility of our system for studying protein-protein interactions in living cells using bimolecular fluorescence complementation. In summary, the established transient expression system provides a powerful tool for investigating protein localization, function, and interactions in C. reinhardtii within a relatively short timeframe, which will significantly facilitate the study of gene function, genome structure, and green biomanufacturing in C. reinhardtii and potentially in other algae.

Effect of Partial Elimination of Mitochondrial DNA on Genome-Wide Identified AOX Gene Family in Chlamydomonas reinhardtii

Using Chlamydomonas as a model organism, we attempted to eliminate mitochondrial DNA (mtDNA) similar to rho0 or rho− cells (completely or partially mtDNA-eliminated cells) in yeast. We successfully generated partially mtDNA-eliminated cells named as crm- cells, causing the inactivation of mitochondrial activity. We used three different chemicals to eliminate mtDNA including acriflavine (AF), ethidium bromide (EB) and dideoxycytidine (ddC) which prevents replication, inhibits POLG (DNA polymerase gamma) and terminates the mtDNA chain, respectively. The qPCR method was used to detect the mtDNA copy number and the selected rrnL6 gene for the detection of mitochondria, as well as the selected Chlamydomonas CC-124 strain. A reduction in the mitochondrial copy number led to a higher expression of AOX1, UCP1, PGRL1 and ICL1, which indicates the disturbance of the mitochondria–chloroplast ATP and NADPH balance. We selected AOX genes to further study this family and carried out a genome-wide search to identify AOX genes in green algae (C. reinhardtii). Our results revealed that C. reinhardtii contains four AOX genes, i.e., CrAOX1, CrAOX2, CrAOX3 and CrAOX4, which are distributed on Chr 3, Chr7 and Chr9. All CrAOX genes were predicted to localize in mitochondria using bioinformatics tools. Phylogenetic analysis suggests that these CrAOXs are subdivided into four groups and genes existing in the same group could perform identical functions. Collinearity analysis describes the strong evolutionary relationships of AOXs between the unicellular green algae Chlamydomonas reinhardtii and the multicellular green algae Volvox carteri. GO (gene ontology) annotation analysis predicted that CrAOXs played an integral part in carrying out alternate oxidative and respirative activities. Three putative miRNAs, cre-miR1162-3p, cre-miR1171 and cre-miR914, targeting the CrAOX2 gene were identified. Our studies have laid a foundation for the further use of partially mtDNA-eliminated cells and elucidating the functional characteristics of the AOX gene family.

Detection and Quantification of Programmed Cell Death in Chlamydomonas reinhardtii: The Example of S-Nitrosoglutathione.

Chlamydomonas (Chlamydomonas reinhardtii) is a unicellular model alga that has been shown to undergo programmed cell death (PCD) that can be triggered in response to different stresses. We have recently shown that Chlamydomonas is particularly well suited to the study and quantification of PCD. We have shown for the first time that S-nitrosoglutathione (GSNO), a nitric oxide (NO) donor, is able to induce PCD and can be used as a study system in Chlamydomonas. In this article, we provide a simple and robust protocol for quantifying GSNO-induced PCD, which can be adapted to any other treatment. We explain how to detect NO production in the cell following GSNO treatment. We show how PCD can be identified simply by analyzing the degradation profile of genomic DNA. We also provide an easy and reproducible cell death quantification protocol, which makes it possible to follow the course of PCD over time and highlight very fine differences in the number of affected cells between different samples. Key features • Use of S-nitrosoglutathione (GSNO) as a means to study programmed cell death (PCD) in Chlamydomonas. • Discrimination of PCD vs. necrosis. • In vivo determination of NO production in the cell. • A simple, robust protocol for PCD quantification.

Diatom abundance in the polar oceans is predicted by genome size.

A principal goal in ecology is to identify the determinants of species abundances in nature. Body size has emerged as a fundamental and repeatable predictor of abundance, with smaller organisms occurring in greater numbers than larger ones. A biogeographic component, known as Bergmann's rule, describes the preponderance, across taxonomic groups, of larger-bodied organisms in colder areas. Although undeniably important, the extent to which body size is the key trait underlying these patterns is unclear. We explored these questions in diatoms, unicellular algae of global importance for their roles in carbon fixation and energy flow through marine food webs. Using a phylogenomic dataset from a single lineage with worldwide distribution, we found that body size (cell volume) was strongly correlated with genome size, which varied by 50-fold across species and was driven by differences in the amount of repetitive DNA. However, directional models identified temperature and genome size, not cell size, as having the greatest influence on maximum population growth rate. A global metabarcoding dataset further identified genome size as a strong predictor of species abundance in the ocean, but only in colder regions at high and low latitudes where diatoms with large genomes dominated, a pattern consistent with Bergmann's rule. Although species abundances are shaped by myriad interacting abiotic and biotic factors, genome size alone was a remarkably strong predictor of abundance. Taken together, these results highlight the cascading cellular and ecological consequences of macroevolutionary changes in an emergent trait, genome size, one of the most fundamental and irreducible properties of an organism.