Penium Margaritaceum Research Articles

Aberrant growth and expansion in Penium margaritaceum triggered by disruption of microtubules and the cell wall.

Penium margaritaceum, a unicellular zygnematophyte (Streptophyta), was employed to elucidate changes in cell expansion when cells were challenged with the fungal pectinolytic enzyme, pectate lyase, and/or the microtubule disrupting agent, amiprophos-methyl (APM). Microtubule disruption by APM results in significant swelling at expansion zones. These swollen zones provide an easy marker for the location of expansion zones, particularly in cells with altered cell wall pectin. Short term treatment with pectate lyase shows pectin degradation primarily at the isthmus expansion zone and two satellite bands, corresponding with the location of future expansion in daughter cells. When the homogalacturonan lattice of the cell wall is removed by treatment with pectate lyase during long treatments, cell division is maintained, but daughter cell products are considerably smaller. Treatment of cells with a mixture of both pectate lyase and APM results in a distinct phenotype, consisting of "dumbbell"-shaped cells, as APM-induced swelling occurs at the novel expansion centers exposed by pectate lyase treatment. These cells also possess other curious alterations including an extensive, chloroplast-free cytoplasmic zone at the center of the cell, a septum containing ß-glycan, arabinogalactan and homogalacturonan epitopes, unique stacks of ER, displaced Golgi bodies and an extensive network of vacuoles. These results provide insight into the importance of cell wall integrity in defining the location of cell growth and division in P. margaritaceum. Understanding these processes in a unicellular zygnematophyte may provide insights into steps involved in the evolution of land plants.

Chemically induced phenotype plasticity in the unicellular zygnematophyte, Penium margaritaceum

Phenotypic plasticity allows a plant cell to alter its structure and function in response to external pressure. This adaptive phenomenon has also been important in the evolution of plants including the emergence of land plants from a streptophyte alga. Penium margaritaceum is a unicellular zygnematophyte (i.e., the group of streptophyte algae that is sister to land plants) that was employed in order to study phenotypic plasticity with a focus on the role of subcellular expansion centers and the cell wall in this process. Live cell fluorescence labeling, immunofluorescence labeling, transmission electron microscopy, and scanning electron microscopy showed significant subcellular changes and alterations to the cell wall. When treated with the actin-perturbing agent, cytochalasin E, cytokinesis is arrested and cells are transformed into pseudo-filaments made of up to eight or more cellular units. When treated with the cyclin-dependent kinase (CDK) inhibitor, roscovitine, cells converted to a unique phenotype with a narrow isthmus zone.

The multifunctional roles of the extracellular matrix in the sessile life of the zygnematophyte Penium margaritaceum: stick, glide and cluster.

Adhesion and consequent adoption of a sessile habit is a common feature of many green algae and was likely a key mechanism in terrestrialization by an ancient zygnematophyte (i.e., the Zygnematophyceae, the group of algae ancestral to land plants). Penium margaritaceum is a unicellular zygnematophyte that exhibits a multistep adhesion mechanism, which leads to the establishment of the sessile habit. Based on microscopic and immunological data, a dense aggregate of fibrils containing arabinogalactan-protein (AGP)-like components covers the cell surface and is responsible for initial adhesion. The AGP-like fibrils are 20 μm in diameter and possess chemical profiles similar to land plant AGPs. The fibrils attach to the inner cell wall layers and are very likely connected to the plasma membrane as glycophosphatidylinositol (GPI) lipid-anchored proteins, as they are susceptible to phospholipase C treatment. The presence of GPI-anchored AGPs in Penium is further supported by the identification of putative Penium homologs of land plant AGP genes responsible for GPI-anchor synthesis. After adhesion, cells secrete a complex heteropolysaccharide-containing extracellular polymeric substance (EPS) that facilitates gliding motility and the formation of cell aggregates. Fucoidan-like polymers, major components of brown algal CWs, are a major constituent of both the EPS and the adhesive layer of the CW and their role in the adhesion process is still to be examined.

Electroporation-based delivery of proteins in Penium margaritaceum and other zygnematophycean algae.

Zygnematophycean algae represent the streptophyte group identified as the closest sister clade to land plants. Their phylogenetic position and growing genomic resources make these freshwater algae attractive models for evolutionary studies in the context of plant terrestrialization. However, available genetic transformation protocols are limited and exclusively DNA-based. To expand the zygnematophycean toolkit, we developed a DNA-free method for protein delivery into intact cells using electroporation. We use confocal microscopy coupled with fluorescence lifetime imaging to assess the delivery of mNeonGreen into algal cells. We optimized the method to obtain high efficiency of delivery and cell recovery after electroporation in two strains of Penium margaritaceum and show that the experimental setup can also be used to deliver proteins in other zygnematophycean species such as Closterium peracerosum-strigosum-littorale complex and Mesotaenium endlicherianum. We discuss the possible applications of this proof-of-concept method.

Endosidin 5 disruption of the Golgi Apparatus and extracellular matrix secretion in the unicellular charophyte, Penium margaritaceum.

Endosidins are a group of small molecular weight compounds, first identified by "chemical biology" screening assays, that have been used to target specific components of the endomembrane system. In this study, we employed multiple microscopy-based screening techniques to elucidate effects of Endosidin 5 (ES5) on the Golgi Apparatus and the secretion of extracellular matrix (ECM) components, Penium margaritaceum. These effects were compared with those caused by treatments with brefeldin A and concanamycin A. Penium margaritaceum's extensive Golgi Apparatus and endomembrane system make it an outstanding model organism for screening changes to the endomembrane system. Here we detail changes to the Golgi Apparatus and secretion of ECM material caused by Endosidin 5. Changes to extracellular polymeric substance (EPS) secretion and cell wall expansion were screened using fluorescence microscopy. Confocal laser scanning microscopy, and transmission electron microscopy were used to assess changes to the Golgi Apparatus, the cell wall and the vesicular network. Electron tomography was also performed in order to detail the changes to the Golgi Apparatus. While other endosidins were able to impact EPS secretion and cell wall expansion only ES5 completely inhibited EPS secretion and cell wall expansion over 24h. Short treatments of ES5 resulted in displacement of the Golgi bodies from their typical linear alignment. The number of cisternae decreased per Golgi stack and trans face cisternae in-curled to form distinct elongate circular profiles. Longer treatment resulted in a transformation of the Golgi body to an irregular aggregate of cisternae. These alterations could be reversed by removal of ES5 and returning cells to culture. ES5 alters secretion of ECM material in Penium by affecting the Golgi apparatus and does so in a markedly different way than other endomembrane inhibitors such as Brefeldin A and Concanamycin A.

Removal of Pb (II) from Aqueous Solution by a Pectin-Producing Alga, Penium margaritaceum, Immobilized on Filter Paper

Lead (Pb) pollution from local mines and industrial use increases risks for human, animal, and plant health. Pectin is an effective chelator of Pb, and it has been shown that a unicellular green alga, Penium margaritaceum, synthesizes pectin in the cell wall. In this study, the ability of P. margaritaceum to remove Pb from an aqueous solution was investigated. Energy dispersive X-ray spectroscopy revealed that two strains of P. margaritaceum accumulated Pb on the cell surface. Hence, P. margaritaceum cells were immobilized on cellulose filter paper. The immobilized algal cells were soaked in 1.0 mg/L Pb solution with gentle shaking for 8 h, and Pb in the solution and on the filter paper was measured by flame atomic absorption spectrometry. The immobilized algal cells continuously decreased the Pb concentration to less than 0.5 mg/L and recovered 31.8–32.7% of added Pb. The specific decrease in Pb and increase in Ca were observed in the presence of 1.0 mg/L each of Ca, Mg, Na, and K. Fourier transform infrared spectra suggested that the carboxylic acid group would be responsible for the adsorption of Pb. This study is the first to demonstrate the effectiveness of the immobilized P. margaritaceum cell in removing Pb from aqueous solutions with simple solid–liquid separation.

The effects of osmotic stress on the cell wall-plasma membrane domains of the unicellular streptophyte, Penium margaritaceum.

Penium margaritaceum is a unicellular zygnematophyte (basal Streptophyteor Charophyte) that has been used as a model organism for the study of cell walls of Streptophytes and for elucidating organismal adaptations that were key in the evolution of land plants.. When Penium is incubated in sorbitol-enhance medium, i.e., hyperosmotic medium, 1000-1500 Hechtian strands form within minutes and connect the plasma membrane to the cell wall. As cells acclimate to this osmotic stress over time, further significant changes occur at the cell wall and plasma membrane domains. The homogalacturonan lattice of the outer cell wall layer is significantly reduced and is accompanied by the formation of a highly elongate, "filamentous" phenotype. Distinct peripheral thickenings appear between the CW and plasma membrane and contain membranous components and a branched granular matrix. Monoclonal antibody labeling of these thickenings indicates the presence of rhamnogalacturonan-I epitopes. Acclimatization also results in the proliferation of the cell's vacuolar networks and macroautophagy. Penium's ability to acclimatize to osmotic stress offers insight into the transition of ancient zygnematophytes from an aquatic to terrestrial existence.

Characterization of Two Zygnema Strains (Zygnema circumcarinatum SAG 698-1a and SAG 698-1b) and a Rapid Method to Estimate Nuclear Genome Size of Zygnematophycean Green Algae.

Zygnematophyceae green algae (ZGA) have been shown to be the closest relatives of land plants. Three nuclear genomes (Spirogloea muscicola, Mesotaenium endlicherianum, and Penium margaritaceum) of ZGA have been recently published, and more genomes are underway. Here we analyzed two Zygnema circumcarinatum strains SAG 698-1a (mating +) and SAG 698-1b (mating −) and found distinct cell sizes and other morphological differences. The molecular identities of the two strains were further investigated by sequencing their 18S rRNA, psaA and rbcL genes. These marker genes of SAG 698-1a were surprisingly much more similar to Z. cylindricum (SAG 698-2) than to SAG 698-1b. Phylogenies of these marker genes also showed that SAG 698-1a and SAG 698-1b were well separated into two different Zygnema clades, where SAG 698-1a was clustered with Z. cylindricum, while SAG 698-1b was clustered with Z. tunetanum. Additionally, physiological parameters like ETRmax values differed between SAG 698-1a and SAG 698-1b after 2 months of cultivation. The de-epoxidation state (DEPS) of the xanthophyll cycle pigments also showed significant differences. Surprisingly, the two strains could not conjugate, and significantly differed in the thickness of the mucilage layer. Additionally, ZGA cell walls are highly enriched with sticky and acidic polysaccharides, and therefore the widely used plant nuclear extraction protocols do not work well in ZGA. Here, we also report a fast and simple method, by mechanical chopping, for efficient nuclear extraction in the two SAG strains. More importantly, the extracted nuclei were further used for nuclear genome size estimation of the two SAG strains by flow cytometry (FC). To confirm the FC result, we have also used other experimental methods for nuclear genome size estimation of the two strains. Interestingly, the two strains were found to have very distinct nuclear genome sizes (313.2 ± 2.0 Mb in SAG 698-1a vs. 63.5 ± 0.5 Mb in SAG 698-1b). Our multiple lines of evidence strongly indicate that SAG 698-1a possibly had been confused with SAG 698-2 prior to 2005, and most likely represents Z. cylindricum or a closely related species.

Tracking the evolutionary innovations of plant terrestrialization.

The gradual transition of the algal ancestor from the freshwater to land has always attracted evolutionary biologists. The recent report of high-quality reference genomes of five Charophyta algae (Spirogloea muscicola, Mesotaenium endlicherianum, Mesostigma viride, Chlorokybus atmophyticus and Penium margaritaceum) and one hornwort (Anthoceros angustus) species sheds light on this fascinating transition. These early diverging plants and algae could have gained new genes from soil bacteria and fungi through horizontal gene transfer (HGT), which was so common during plant terrestrialization and may outrun our expectations. Through reviewing and critical thinking about the advancements on these plant genomes, here, I propose three prospective research directions that need to address in the future: (i) due to the ubiquitous nature of viruses that is similar to soil bacteria and fungi, there is less attention to viruses that probably also play an important role in the genome evolution of plants via HGT; (ii) multicellularity has occurred many times independently, but we still know a little about the biological and ecological mechanisms leading to multi-cellularity in Streptophyta; (iii) and most importantly, the quantitative relationships between genetic innovations and environmental variables such as temperature, precipitation and solar radiation, need pioneering research collaborated by biological evolutionists, computer scientists, and ecologists, which are crucial for understanding the macroevolution of plants and could also be used to simulate the evolution of plants under future climate change.

First person – Destiny Davis

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Destiny Davis is first author on ‘Callose deposition is essential for the completion of cytokinesis in the unicellular alga Penium margaritaceum’, published in JCS. Destiny conducted the research described in this article while a PhD student in Georgia Drakakaki's lab at University of California, Davis, CA, USA. She is now a postdoc in the lab of Jenny Mortimer at the Joint BioEnergy Institute (JBEI), Emeryville, CA, USA, where she is endlessly fascinated by the inner workings of the plant cell related to the cell wall.

Experimental Manipulation of Pectin Architecture in the Cell Wall of the Unicellular Charophyte, Penium Margaritaceum.

Pectins represent one of the main components of the plant primary cell wall. These polymers have critical roles in cell expansion, cell-cell adhesion and response to biotic stress. We present a comprehensive screening of pectin architecture of the unicellular streptophyte, Penium margaritaceum. Penium possesses a distinct cell wall whose outer layer consists of a lattice of pectin-rich fibers and projections. In this study, cells were exposed to a variety of physical, chemical and enzymatic treatments that directly affect the cell wall, especially the pectin lattice. Correlative analyses of pectin lattice perturbation using field emission scanning electron microscopy, confocal laser scanning microscopy, and transmission electron microscopy demonstrate that pectin lattice microarchitecture is both highly sensitive and malleable.

Addition to the desmid flora (Charophyta: Desmidiales) of Nizhnevartovsk district, KMAO-Yugra, Russia

Five species of desmid algae new to Nizhnevartovsk city are reported. Three species (Closterium abruptum,Cl. jenneri, Euastrum ansatum) are rare for Nizhnevartovsk administrative region, one (Penium margaritaceum) is newfor Khanty-Mansi Autonomous Okrug – Yugra.

The Penium margaritaceum Genome: Hallmarks of the Origins of Land Plants

The evolutionary features and molecular innovations that enabled plants to first colonize land are not well understood. Here, insights are provided through our report of the genome sequence of the unicellular alga Penium margaritaceum, a member of the Zygnematophyceae, the sister lineage to land plants. The genome has a high proportion of repeat sequences that are associated with massive segmental gene duplications, likely facilitating neofunctionalization. Compared with representatives of earlier diverging algal lineages, P.margaritaceum has expanded repertoires of gene families, signaling networks, and adaptive responses that highlight the evolutionary trajectory toward terrestrialization. These encompass a broad range of physiological processes and protective cellular features, such as flavonoid compounds and large families of modifying enzymes involved in cell wall biosynthesis, assembly, and remodeling. Transcriptome profiling further elucidated adaptations, responses, and selective pressures associated with the semi-terrestrial ecosystems of P.margaritaceum, where a simple body plan would be an advantage.

Endomembrane architecture and dynamics during secretion of the extracellular matrix of the unicellular charophyte, Penium margaritaceum.

The extracellular matrix (ECM) of many charophytes, the assemblage of green algae that are the sister group to land plants, is complex, produced in large amounts, and has multiple essential functions. An extensive secretory apparatus and endomembrane system are presumably needed to synthesize and secrete the ECM, but structural details of such a system have not been fully characterized. Penium margaritaceum is a valuable unicellular model charophyte for studying secretion dynamics. We report that Penium has a highly organized endomembrane system, consisting of 150–200 non-mobile Golgi bodies that process and package ECM components into different sets of vesicles that traffic to the cortical cytoplasm, where they are transported around the cell by cytoplasmic streaming. At either fixed or transient areas, specific cytoplasmic vesicles fuse with the plasma membrane and secrete their constituents. Extracellular polysaccharide (EPS) production was observed to occur in one location of the Golgi body and sometimes in unique Golgi hybrids. Treatment of cells with brefeldin A caused disruption of the Golgi body, and inhibition of EPS secretion and cell wall expansion. The structure of the endomembrane system in Penium provides mechanistic insights into how extant charophytes generate large quantities of ECM, which in their ancestors facilitated the colonization of land.

Callose deposition is essential for the completion of cytokinesis in the unicellular alga Penium margaritaceum.

Cytokinesis in land plants involves the formation of a cell plate that develops into the new cell wall. Callose, a β-1,3 glucan, accumulates at later stages of cell plate development, presumably to stabilize this delicate membrane network during expansion. Cytokinetic callose is considered specific to multicellular plant species, because it has not been detected in unicellular algae. Here we present callose at the cytokinesis junction of the unicellular charophyte, Penium margaritaceum Callose deposition at the division plane of P. margaritaceum showed distinct, spatiotemporal patterns likely representing distinct roles of this polymer in cytokinesis. Pharmacological inhibition of callose deposition by endosidin 7 resulted in cytokinesis defects, consistent with the essential role for this polymer in P. margaritaceum cell division. Cell wall deposition at the isthmus zone was also affected by the absence of callose, demonstrating the dynamic nature of new wall assembly in P. margaritaceum The identification of candidate callose synthase genes provides molecular evidence for callose biosynthesis in P. margaritaceum The evolutionary implications of cytokinetic callose in this unicellular zygnematopycean alga is discussed in the context of the conquest of land by plants.This article has an associated First Person interview with the first author of the paper.

Protoplast Isolation and Manipulation in the Unicellular Model Plant Penium margaritaceum.

The unicellular freshwater green alga Penium margaritaceum has become a novel and valuable model organism for elucidating cell wall dynamics in plants. We describe a rapid and simple means for isolating protoplasts using commercial enzymes in a mannitol-based buffer. Protoplasts can be cultured and cell wall recovery can be monitored in sequentially diluted mannitol-based medium. We also describe an optimized protocol to prepare highly pure, organelle-free nuclei fractions from protoplasts using sucrose gradients. This technology provides a new and effective tool in Penium biology that can be used for analysis of cell wall polymer deposition, organelle isolation and characterization, and molecular research including genetic transformation and somatic hybridization.

The Secretome and N-Glycosylation Profiles of the Charophycean Green Alga, Penium margaritaceum, Resemble Those of Embryophytes.

The secretome can be defined as the population of proteins that are secreted into the extracellular environment. Many proteins that are secreted by eukaryotes are N-glycosylated. However, there are striking differences in the diversity and conservation of N-glycosylation patterns between taxa. For example, the secretome and N-glycosylation structures differ between land plants and chlorophyte green algae, but it is not clear when this divergence took place during plant evolution. A potentially valuable system to study this issue is provided by the charophycean green algae (CGA), which is the immediate ancestors of land plants. In this study, we used lectin affinity chromatography (LAC) coupled with mass spectrometry to characterize the secretome including secreted N-glycoproteins of Penium margaritaceum, which is a member of the CGA. The identified secreted proteins and N-glycans were compared to those known from the chlorophyte green alga Chlamydomonas reinhardtii and the model land plant, Arabidopsis thaliana, to establish their evolutionary context. Our approach allowed the identification of cell wall proteins and proteins modified with N-glycans that are identical to those of embryophytes, which suggests that the P. margaritaceum secretome is more closely related to those of land plants than to those of chlorophytes. The results of this study support the hypothesis that many of the proteins associated with plant cell wall modification as well as other extracellular processes evolved prior to the colonization of terrestrial habitats.

Isolation and manipulation of protoplasts from the unicellular green alga Penium margaritaceum

BackgroundThe unicellular charophycean green alga Penium margaritaceum has emerged as an appealing experimental organism in plant cell wall and cell biology research. Innovative practical approaches in the manipulation and maintenance of this unicellular model alga are needed in order to probe the complexities of its subcellular and molecular machinery. Protoplast isolation and manipulation expedites a new range of experimental possibilities for Penium-based studies. These include enhanced means of isolation of subcellular components and macromolecules, application of intracellular probes for high resolution microscopy of live cells, transformation studies and analysis of the fundamental mechanisms of plant cell expansion and wall polymer deposition.ResultsWe present a methodology for enzyme-based digestion of the Penium cell wall and the isolation of protoplasts. The subcellular events associated with this technology are presented using multiple microscopy-based techniques. We also provide protocols for applying an array of intracellular dyes that can be used as markers for specific organelles and membrane microdomains in live cells. Finally, we present a protocol for the purification of a nuclei-rich fraction from protoplasts, which can be used for the isolation of nuclear DNA.ConclusionProtoplast isolation, culturing and manipulation provide valuable means for molecular and cellular studies of Penium. The protocol described here offers a rapid and effective mechanism for fast and effective production of protoplasts. Subsequently, the protoplasts may be used for microscopy-based studies of specific subcellular components and the isolation of organelles and nuclear DNA. These methods offer a new practical methodology for future studies of this model organism in cell and molecular biology.

Imaging the Dynamics of Cell Wall Polymer Deposition in the Unicellular Model Plant, Penium margaritaceum.

The unicellular green alga, Penium margaritaceum, represents a novel and valuable model organism for elucidating cell wall dynamics in plants. This organism's cell wall contains several polymers that are highly similar to those found in the primary cell walls of land plants. Penium is easily grown in laboratory culture and is effectively manipulated in various experimental protocols including microplate assays and correlative microscopy. Most importantly, Penium can be live labeled with cell wall-specific antibodies or other probes and returned to culture where specific cell wall developmental events can be monitored. Additionally, live cells can be rapidly cryo-fixed and cell wall surface microarchitecture can be observed with variable pressure scanning electron microscopy. Here, we describe the methodology for maintaining Penium for experimental cell wall enzyme studies.

UNVEILING ALGAL BIODIVERSITY OF EL-FARAFRA OASIS (WESTERN DESERT, EGYPT) AND POTENTIAL RELEVANCE OF ITS USE IN WATER BIO-ASSESSMENT: SPECIAL INTEREST ON SPRINGS AND DRILLED WELLS

For the first time, comprehensive phycological screening on El-Farafra Oasis (Western Desert, Egypt) had been implemented for unveiling and characterization of its cryptic algal diversification, with special interest on springs and drilled wells. Overall during summer 2011 and winter 2012, 82 different algal taxa have been found in all studied habitats as naturally-grown and culture-induced propagations. Of these, different 14 algal strains have been recorded only using water-culturing techniques on different four algal media (Kolkwitz's solution, Chu #10, BBM and modified BG-11 media). Culturing techniques not only permitted to discover the cryptic algal taxa present in the viable spores state, but also allowed to follow and confirm the complete life cycle of the rare stigonematalean true T-branched cyanoprokaryote Westiellopsis prolifica. This inventory enriched the freshwater Egyptian algal flora with 6 new algal taxa for the first time namely Symploca dubia, Westiellopsis prolifica, Euastrum insulare, Penium margaritaceum, Zygnemopsis sp. and Nitellopsis obtusa. In this study, some recorded taxa are considered to be crenophilous, e.g. Geitlerinema jasorvense and Phormidium carboniciphilum, to be crenoxenic e.g. Jaaginema subtilissimum, Phormidium corium, Westiellopsis prolifica and Zygnemopsis sp. Only, Oscillatoria tenuis had the highest-grade constancy of occurrence throughout the whole investigation period. This species shows a strong α-mesosaprobic indication for water eutrophication in this oasis. In general, El-Farafra Oasis wells and springs are considered isolated and azonal habitats still bearing partially near-natural conditions with some agricultural, pastures and humans impacts, and have freshwater type with slight seasonal variations in their mineralization contents as a kind of ecotones-characteristic features. Finally, these hyper-arid-desert-dwelling water systems are just about excellent potable water resources in the context of water quality guidelines of Egyptian Higher Committee for Water (EHCW) and World Health Organization (WHO), but they substantially still need special governmental efforts in the future to mainly eliminate the relatively high contents of Fe, Mn and Zn.