Diversity of genome size and chromosome number in homothallic and heterothallic strains of the Closterium peracerosum-strigosum-littorale complex (Desmidiales, Zygnematophyceae, Streptophyta).

The evolutionary transitions of mating systems between outcrossing and self-fertilization are often suggested to associate with the cytological and genomic changes, but the empirical reports are limited in multicellular organisms. Here we used the unicellular zygnematophycean algae, the Closterium peracerosum-strigosum-littorale (C. psl.) complex, to address whether genomic properties such as genome sizes and chromosome numbers are associated with mating system transitions between homothallism (self-fertility) and heterothallism (self-sterility). Phylogenetic analysis revealed the polyphyly of homothallic strains, suggesting multiple transitions between homothallism and heterothallism in the C. psl. complex. Flow cytometry analysis identified a more than 2-fold genome size variation, ranging from 0.53 to 1.42 Gbp, which was positively correlated with chromosome number variation between strains. Although we did not find consistent trends in genome size change and mating system transitions, the mean chromosome sizes tend to be smaller in homothallic strains than in their relative heterothallic strains. This result suggests that homothallic strains possibly have more fragmented chromosomes, which is consistent with the argument that self-fertilizing populations may tolerate more chromosomal rearrangements.

Extensive Copy Number Variation Explains Genome Size Variation in the Unicellular Zygnematophycean Alga, Closterium peracerosum-strigosum-littorale Complex.

Genome sizes are known to vary within and among closely related species, but the knowledge about genomic factors contributing to the variation and their impacts on gene functions is limited to only a small number of species. This study identified a more than 2-fold heritable genome size variation among the unicellular Zygnematophycean alga, Closterium peracerosum-strigosum-littorale (C. psl.) complex, based on short-read sequencing analysis of 22 natural strains and F1 segregation analysis. Six de novo assembled genomes revealed that genome size variation is largely attributable to genome-wide copy number variation (CNV) among strains rather than mating type-linked genomic regions or specific repeat sequences such as rDNA. Notably, about 30% of genes showed CNV even between strains that can mate with each other. Transcriptome and gene ontology analysis demonstrated that CNV is distributed nonrandomly in terms of gene functions, such that CNV was more often observed in the gene set with stage-specific expression. Furthermore, in about 30% of these genes with CNV, the expression level does not increase proportionally with the gene copy number, suggesting presence of dosage compensation, which was overrepresented in genes involved in basic biological functions, such as translation. Nonrandom patterns in gene duplications and corresponding expression changes in terms of gene functions may contribute to maintaining the high level of CNV associated with extensive genome size variation in the C. psl. complex, despite its possible detrimental effects.

Molecular cloning and oxidative-stress responses of a novel Phi class glutathione S-transferase (GSTF) gene in the freshwater algae Closterium ehrenbergii.

Glutathione S-transferases (GSTs) belong to a family of enzymes involved in diverse biological processes, including detoxification and protection against oxidative damage. Here, we determined the full-length sequence (915 bp) of a novel Phi class cytosolic glutathione S-transferase (GSTF) gene from the green algae Closterium ehrenbergii. We examined the gene structure and expression patterns in response to metals and endocrine disrupting chemicals (EDCs). It was significantly upregulated by metals, but responded differently to EDCs. The highest up-regulation of CeGSTF was registered under 0.1 mg/L CuCl2 and 0.01 mg/L CuSO4 treatments. In a 72-h course experiment with treatment of 0.1 mg/L CuCl2 , CeGSTF was dramatically induced at 6 h, and then gradually decreased with increasing exposure time. This was consistent with the increase in both GST activity and ROS production in copper-treated cells. These results suggest that CeGSTF may be involved in detoxification mechanisms associated with oxidative stress in green algae.

Molecular cloning of heat shock protein 70 and HOP from the freshwater green algae Closterium ehrenbergii and their responses to stress.

Heat shock proteins (HSPs) and HSP70-HSP90 organizing proteins (HOPs) are related, and they function together to maintain cellular homeostasis and respond to stress. In the present study, we reported the first molecular characteristics of HSP70 (designated as CeHSP70) and HOP (designated as CeHOP) genes from the freshwater green algae Closterium ehrenbergii and examined the changes in their expression profiles under heat stress and toxic chemicals treatment. CeHSP70 presented the conserved motif patterns and EEVD domain specific to cytosolic HSP70; CeHOP contained a typical domain of TPR repeats. Real-time PCR analysis showed that thermal stress considerably up-regulated both CeHOP and CeHSP70. In addition, the genes were significantly induced by CuCl2, CuSO4, and NiSO4, but not by K2Cr2O7, herbicide, and endocrine disrupting chemicals. These results suggest that CeHOP and CeHSP70 function together and play a role in responses to specific stressors and indicate their possible use as sensitive specific biomarkers in risk assessments.

Molecular identification, differential expression and protective roles of iron/manganese superoxide dismutases in the green algae Closterium ehrenbergii against metal stress.

The green microalgae Closterium ehrenbergii is an ideal organism for ecotoxicology assessments; however, its toxicogenomics has been insufficiently examined. Here, we identified three iron/manganese superoxide dismutase (SOD) genes (designated as CeFeSOD1, CeFeSOD2, and CeMnSOD) from C. ehrenbergii and examined their expressional patterns for four metals (iron, manganese, copper, and nickel). These genes encoded 362, 224, and 245 amino acids, respectively; signal-peptide analysis showed that they were differentially located in chloroplasts, cytosol, or mitochondria. Real-time PCRs revealed differential expression patterns according to metal and doses. Interestingly, CeSODs displayed no noticeable changes to treatment with their corresponding cofactor metals, iron or manganese, even at high doses. However, they were obviously up-regulated under toxic metal (copper and nickel) exposure, exhibiting approximately 10.8- and 4.4-fold increases, respectively. Copper (0.2 mg/L) dramatically stimulated intracellular reactive oxygen species (ROS) formation, increased SOD activity, and reduced photosynthetic efficiency in C. ehrenbergii. These results suggest that CeFeSODs and CeMnSOD might be involved in protecting cells against damage and oxidative stress caused by non-cofactor metals, such as copper and nickel. These genes were sensitively responsive at levels well below the EC50, showing that they can be used as molecular biomarkers to assess the toxicity of specific metal contaminants.

De novo transcriptome of the diatom Cylindrotheca closterium identifies genes involved in the metabolism of anti-inflammatory compounds.

Diatoms are the most diverse and abundant group of phytoplankton species and represent a huge reservoir of marine natural products with possible application for human health. Several diatoms are known to have anticancer, anti-inflammatory, antioxidant and anti-microbial properties, but the compounds responsible of these activities are often still unknown. The diatom Cylindrotheca closterium showed anti-inflammatory properties inhibiting TNFα release in human monocytic leukemia cells. In this study, we present the full transcriptome of C. closterium, and used an -omic approach to identify transcripts coding enzymes that can be involved in the synthesis/degradation of anti-inflammatory compounds. This approach allowed to identify phosphatidylinositol-3-phosphatase, phosphatidylinositol 3-kinase catalytic subunit type 3, phosphatidylinositol N-acetylglucosaminyltransferase subunit A, monogalactosyldiacylglycerol synthase and violaxanthin de-epoxidase, which are known to be involved in anti-inflammatory compound metabolism. When C. closterium was cultured in silica-starvation conditions, selected as stress condition to potentially trigger the synthesis of bioactive metabolites, anti-inflammatory activity was lost and expression levels of the analyzed transcripts were reduced. These data suggested that the control culturing condition was the most active. This study used for the first time a transcriptomic-guided approach to identify enzymes involved in anti-inflammatory compound metabolism, directing future discoveries of marine natural products in microalgae.

Small heat shock protein genes of the green algae Closterium ehrenbergii: Cloning and differential expression under heat and heavy metal stresses.

The freshwater green algae Closterium ehrenbergii has been considered as a model for eco-toxicological assessment in aquatic systems. Heat shock proteins (HSPs) are a class of highly conserved proteins produced in all living organisms, which participate in environmental stress responses. In the present study, we determined the cDNA sequences of small heat shock protein 10 (sHSP10) and sHSP17.1 from C. ehrenbergii, and examined the physiological changes and transcriptional responses of the genes after exposure to thermal shock and toxicants treatments. The open reading frame (ORF) of CeHSP10 was 300 bp long, encoding 99 amino acid (aa) residues (10.53 kDa) with a GroES chaperonin conserved site of 22 aa. The CeHSP17.1 had a 468 bp ORF, encoding 155 aa with a conserved C-terminal α-crystallin domain. For heat stress, cells presented pigment loss and possible chloroplast damage, with an up-regulation in the expression of both sHSP10 and sHSP17.1 genes. As for the heavy metal stressors, an increase in the production of reactive oxygen species was registered in a dose dependent manner, with a significant up-regulation of both sHSP10 and sHSP17.1 genes. These results suggest that sHSP genes in C. ehrenbergii may play a role in responses to stress environments, and they could be used as an early detection parameter as biomarker genes in molecular toxicity assessments.

Identification of a new mating group and reproductive isolation in the Closterium peracerosum-strigosum-littorale complex.

Reproductive isolation is essential for the process of speciation. In order to understand speciation, it is necessary to compare one mating group with other phylogenetically related but reproductively isolated groups. The Closterium peracerosum-strigosum-littorale (C. psl.) complex is a unicellular isogamous zygnematophycean alga, which is believed to share a close phylogenetic relationship with the land plants. In this study, we identified a new mating group, named group G, of C. psl. complex and compared its physiological and biochemical characteristics with the mating group I-E, which was closely related to the mating group G. Zygospores are typically formed as a result of conjugation between mating-type plus (mt+) and mating-type minus (mt-) cells in the same mating group during sexual reproduction. Crossing experiments revealed mating groups G and I-E were reproductively isolated from each other, but the release of lone protoplasts from mt- cells of mating group G was induced in the presence of mt+ cells of mating group I-E. In fact, the sex pheromone, protoplast-release-inducing protein of mating group I-E induced the release of protoplasts from mt- cells of mating group G. When mt+ and mt- cells of both mating groups I-E and G were co-cultured (multiple-choice matings), the zygospore formation of mating group G, but not that of mating group I-E, was inhibited. Based on these results, we propose a possible mechanism of reproductive isolation between the two mating groups and suggest the presence of sexual interference between mating group G and mating group I-E.

CRISPR/Cas9-based knockouts reveal that CpRLP1 is a negative regulator of the sex pheromone PR-IP in the Closterium peracerosum-strigosum-littorale complex.

Heterothallic strains of the Closterium peracerosum-strigosum-littorale (C. psl.) complex have two sexes, mating-type plus (mt+) and mating-type minus (mt-). Conjugation between these two sexes is regulated by two sex pheromones, protoplast-release-inducing protein (PR-IP) and PR-IP Inducer, which are produced by mt+ and mt- cells, respectively. PR-IP mediates the release of protoplasts from mt- cells during mating. In this study, we examined the mechanism of action of CpRLP1 (receptor-like protein 1), which was previously identified in a cDNA microarray analysis as one of the PR-IP-inducible genes. Using CRISPR/Cas9 technology, we generated CpRLP1 knockout mutants in mt- cells of the C. psl. complex. When the knockout mt- cells were mixed with wild-type mt+ cells, conjugation was severely reduced. Many cells released protoplasts without pairing, suggesting a loss of synchronization between the two mating partners. Furthermore, the knockout mutants were hypersensitive to PR-IP. We conclude that CpRLP1 is a negative regulator of PR-IP that regulates the timing of protoplast release in conjugating C. psl. cells. As the first report of successful gene knockout in the class Charophyceae, this study provides a basis for research aimed at understanding the ancestral roles of genes that are indispensable for the development of land plants.

A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites.

The human gut microbiota produces dozens of metabolites that accumulate in the bloodstream, where they can have systemic effects on the host. Although these small molecules commonly reach concentrations similar to those achieved by pharmaceutical agents, remarkably little is known about the microbial metabolic pathways that produce them. Here we use a combination of genetics and metabolic profiling to characterize a pathway from the gut symbiont Clostridium sporogenes that generates aromatic amino acid metabolites. Our results reveal that this pathway produces twelve compounds, nine of which are known to accumulate in host serum. All three aromatic amino acids (tryptophan, phenylalanine and tyrosine) serve as substrates for the pathway, and it involves branching and alternative reductases for specific intermediates. By genetically manipulating C. sporogenes, we modulate serum levels of these metabolites in gnotobiotic mice, and show that in turn this affects intestinal permeability and systemic immunity. This work has the potential to provide the basis of a systematic effort to engineer the molecular output of the gut bacterial community.

Sexual reproduction and sex determination in green algae.

The sexual reproductive processes of some representative freshwater green algae are reviewed. Chlamydomonas reinhardtii is a unicellular volvocine alga having two mating types: mating type plus (mt+) and mating type minus (mt-), which are controlled by a single, complex mating-type locus. Sexual adhesion between the gametes is mediated by sex-specific agglutinin molecules on their flagellar membranes. Cell fusion is initiated by an adhesive interaction between the mt+ and mt- mating structures, followed by localized membrane fusion. The loci of sex-limited genes and the conformation of sex-determining regions have been rearranged during the evolution of volvocine algae; however, the essential function of the sex-determining genes of the isogamous unicellular Chlamydomonas reinhardtii is conserved in the multicellular oogamous Volvox carteri. The sexual reproduction of the unicellular charophycean alga, Closterium peracerosum-strigosum-littorale complex, is also focused on here. The sexual reproductive processes of heterothallic strains are controlled by two multifunctional sex pheromones, PR-IP and PR-IP Inducer, which independently promote multiple steps in conjugation at the appropriate times through different induction mechanisms. The molecules involved in sexual reproduction and sex determination have also been characterized.

Algal ancestor of land plants was preadapted for symbiosis.

Colonization of land by plants was a major transition on Earth, but the developmental and genetic innovations required for this transition remain unknown. Physiological studies and the fossil record strongly suggest that the ability of the first land plants to form symbiotic associations with beneficial fungi was one of these critical innovations. In angiosperms, genes required for the perception and transduction of diffusible fungal signals for root colonization and for nutrient exchange have been characterized. However, the origin of these genes and their potential correlation with land colonization remain elusive. A comprehensive phylogenetic analysis of 259 transcriptomes and 10 green algal and basal land plant genomes, coupled with the characterization of the evolutionary path leading to the appearance of a key regulator, a calcium- and calmodulin-dependent protein kinase, showed that the symbiotic signaling pathway predated the first land plants. In contrast, downstream genes required for root colonization and their specific expression pattern probably appeared subsequent to the colonization of land. We conclude that the most recent common ancestor of extant land plants and green algae was preadapted for symbiotic associations. Subsequent improvement of this precursor stage in early land plants through rounds of gene duplication led to the acquisition of additional pathways and the ability to form a fully functional arbuscular mycorrhizal symbiosis.

A Receptor-Like Kinase, Related to Cell Wall Sensor of Higher Plants, is Required for Sexual Reproduction in the Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex.

Here, we cloned the CpRLK1 gene, which encodes a receptor-like protein kinase expressed during sexual reproduction, from the heterothallic Closterium peracerosum-strigosum-littorale complex, one of the closest unicellular alga to land plants. Mating-type plus (mt(+)) cells with knockdown of CpRLK1 showed reduced competence for sexual reproduction and formed an abnormally enlarged conjugation papilla after pairing with mt(-) cells. The knockdown cells were unable to release a naked gamete, which is indispensable for zygote formation. We suggest that the CpRLK1 protein is an ancient cell wall sensor that now functions to regulate osmotic pressure in the cell to allow proper gamete release.

Evaluation and selection of reference genes for ecotoxicogenomic study of the green alga Closterium ehrenbergii using quantitative real-time PCR.

The green alga Closterium ehrenbergii occurs in fresh water environments and has been suggested as a model for ecotoxicological assessment. Quantitative real-time PCR (qRT-PCR), with its high sensitivity and specificity, is a preferred method for reliable quantification of gene expression levels. qRT-PCR requires reference genes to normalize the transcription level of the target gene, and selection of appropriate references is crucial. Here, we evaluated nine housekeeping genes, that is, 18S rRNA, ACT, TUA, TUB, eIF, H4, UBQ, rps4, and GAPDH, using 34 RNA samples of C. ehrenbergii cultured in various environments (e.g. exposure to heat shock, UV, metals, and non-metallic chemicals). Each housekeeping gene tested displayed different ranges of C T values for each experimental condition. The gene stability was determined using the descriptive statistic software geNorm, which showed that ACT, H4, and TUA were the most suitable reference genes for all the conditions tested. In addition, at least three genes were required for proper normalization. With these references, we assessed the expression level of the heat shock protein 70 (HSP70) gene in C. ehrenbergii cells exposed to thermal and toxic contaminant stress and found that it was significantly up-regulated by these stressors. This study provides potential reference genes for gene expression studies on C. ehrenbergii with qRT-PCR.

Zygospore formation between homothallic and heterothallic strains of Closterium.

Zygospore formation in different strains of the Closterium peracerosum-strigosum-littorale complex was examined in this unicellular isogamous charophycean alga to shed light on gametic mating strains in this taxon, which is believed to share a close phylogenetic relationship with land plants. Zygospores typically form as a result of conjugation between mating-type plus (mt(+)) and mating-type minus (mt(-)) cells during sexual reproduction in the heterothallic strain, similar to Chlamydomonas. However, within clonal cells, zygospores are formed within homothallic strains, and the majority of these zygospores originate as a result of conjugation of two recently divided sister gametangial cells derived from one vegetative cell. In this study, we analyzed conjugation of homothallic cells in the presence of phylogenetically closely related heterothallic cells to characterize the reproductive function of homothallic sister gametangial cells. The relative ratio of non-sister zygospores to sister zygospores increased in the presence of heterothallic mt(+) cells, compared with that in the homothallic strain alone and in a coculture with mt(-) cells. Heterothallic cells were surface labeled with calcofluor white, permitting fusions with homothallic cells to be identified and confirming the formation of hybrid zygospores between the homothallic cells and heterothallic mt(+) cells. These results show that at least some of the homothallic gametangial cells possess heterothallic mt(-)-like characters. This finding supports speculation that division of one vegetative cell into two sister gametangial cells is a segregative process capable of producing complementary mating types.

Stable nuclear transformation of the Closterium peracerosum-strigosum-littorale complex.

Although charophycean algae form a relevant monophyly with embryophytes and hence occupy a fundamental place in the development of Streptophyta, no tools for genetic transformation in these organisms have been developed. Here we present the first stable nuclear transformation system for the unicellular Zygnematales, the Closterium peracerosum-strigosum-littorale complex (C. psl complex), which is one of the most useful organisms for experimental research on charophycean algae. When a vector, pSA106, containing the dominant selectable marker ble (phleomycin-resistant) gene and a reporter cgfp (Chlamydomonas-adapted green fluorescent protein) gene was introduced into cells via particle bombardment, a total of 19 phleomycin-resistant cells were obtained in the presence of a low concentration of phleomycin. Six isogenic strains isolated using conditioned medium showed consecutive cgfp expression and long-term stability for phleomycin resistance. DNA analyses verified single or tandem/redundant integration of ~10 copies of pSA106 into the C. psl complex genome. We also constructed an overexpression vector, pSA1102, and then integrated a CpPI gene encoding minus-specific sex pheromone into pSA1102. Ectopic overexpression of CpPI and the pheromonal function were confirmed when the vector pSA1102_CpPI was introduced into mt(+) cells. The present efficient transformation system for the C. psl complex should provide not only a basis for molecular investigation of Closterium but also an insight into important processes in early development and evolution of Streptophyta.

Characterization and molecular cloning of conjugation-regulating sex pheromones in homothallic Closterium.

Conjugation-regulating pheromones were analyzed in homothallic Closterium for the first time. Members of the Closterium peracerosum-strigosum-littorale complex are unicellular charophycean algae in which there are two modes of zygospore formation: heterothallism and homothallism. A homothallic strain of Closterium (designation, kodama20) forms selfing zygospores via the conjugation of two sister gametangial cells derived from one vegetative cell. Conjugation-promoting and -suppressing activities, against cells at very low (1 x 10(2) cells ml(-1)) and normal (1 x 10(4) cells ml(-1)) cell density, respectively, were detected in the medium in which cells of a normal density had been cultured. Pheromone activities decreased to 20% after incubation at 60 °C for 10 min. The release and action of the pheromones was dependent on light. The culture medium was subjected to gel filtration, and both active substances had an apparent molecular mass of 17 kDa; this was similar to that previously reported for the heterothallic sex-specific pheromone protoplast-release-inducing protein (PR-IP) Inducer. cDNAs encoding the orthologs of PR-IP Inducer were isolated from the homothallic strain. Recombinant PR-IP Inducers produced by yeast cells showed conjugation-promoting activity. These results indicate that conjugation of the homothallic strain is regulated by an ortholog of a heterothallic sex-specific pheromone.