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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.

The plant endomembrane system facilitates the transport of polysaccharides, associated enzymes, and glycoproteins through its dynamic pathways. Although enzymes involved in cell wall biosynthesis have been identified, little is known about the endomembrane-based transport of glycan components. This is partially attributed to technical challenges in biochemically determining polysaccharide cargo in specific vesicles. Here, we introduce a hybrid approach addressing this limitation. By combining vesicle isolation with a large-scale carbohydrate antibody arraying technique, we charted an initial large-scale map describing the glycome profile of the SYNTAXIN OF PLANTS61 (SYP61) trans-Golgi network compartment in Arabidopsis (Arabidopsis thaliana). A library of antibodies recognizing specific noncellulosic carbohydrate epitopes allowed us to identify a range of diverse glycans, including pectins, xyloglucans (XyGs), and arabinogalactan proteins in isolated vesicles. Changes in XyG- and pectin-specific epitopes in the cell wall of an Arabidopsis SYP61 mutant corroborate our findings. Our data provide evidence that SYP61 vesicles are involved in the transport and deposition of structural polysaccharides and glycoproteins. Adaptation of our methodology can enable studies characterizing the glycome profiles of various vesicle populations in plant and animal systems and their respective roles in glycan transport defined by subcellular markers, developmental stages, or environmental stimuli.

The deposition and modulation of constituent polymers of plant cell walls are profoundly important events during plant development. Identification of specific polymers within assembled walls during morphogenesis and in response to stress conditions represents a major goal of plant cell biologists. Arabidopsis thaliana is a model organism that has become central to research focused on fundamental plant processes including those related to plant wall dynamics. Its fast life cycle and easy access to a variety of mutants and ecotypes of Arabidopsis have stimulated the need for rapid assessment tools to probe its wall organization at the cellular and subcellular levels. We describe two rapid assessment techniques that allow for elucidation of the cell wall polymers of root hairs and high-resolution analysis of surface features of various vegetative organs. Live organism immunolabeling of cell wall polymers employing light microscopy and confocal laser scanning microscopy can be effectively performed using a large microplate-based screening strategy (see Figs. 1 and 2). Rapid cryofixation and imaging of variable pressure scanning electron microscopy also allows for imaging of surface features of all portions of the plant as clearly seen in Fig. 3.

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.

Cytokinesis in land plants involves the formation of a cell plate that develops into the new cell wall. Callose is a β-1,3 glucan that transiently accumulates at later stages of cell plate development and is thought to stabilize the delicate membrane network of the cell plate as it expands. Cytokinetic callose deposition is currently considered specific to multicellular plant species as it has not been detected in unicellular algae. Here we present callose at the cytokinesis junction of the unicellular charophyte,Penium margaritaceum. Notably, callose deposition at the division plane ofP. margaritaceumshowed distinct, spatiotemporal patterns that could represent distinct roles of this polymer in cytokinesis and cell wall assembly. Pharmacological inhibition of cytokinetic callose deposition by Endosidin 7 treatment resulted in cytokinesis defects, consistent with the essential role for this polymer inP. margaritaceumcell division. Cell wall deposition and assembly at the isthmus zone was also affected by the absence of callose, demonstrating the dynamic nature of new wall assembly inP. margaritaceum. The identification of candidate callose synthase genes provides molecular evidence for callose biosynthesis inP. margaritaceum. The evolutionary implications of cytokinetic callose in this unicellular Zygnematopycean alga is discussed in the context of the conquest of land by plants.Summary StatementEvolutionarily conserved callose inPenium margaritaceumis essential for the completion of cytokinesis.

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

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 deposition and modulation of constituent polymers of plant cell walls are profoundly important events during plant development. Identification of specific polymers within assembled walls during morphogenesis and in response to stress conditions represents a major goal of plant cell biologists. Arabidopsis thaliana is a model organism that has become central to research focused on fundamental plant processes including those related to plant wall dynamics. Its fast life cycle and easy access to a variety of mutants and ecotypes of Arabidopsis have stimulated the need for rapid assessment tools to probe its wall organization at the cellular and subcellular levels. We describe two rapid assessment techniques that allow for elucidation of the cell wall polymers of root hairs and high-resolution analysis of surface features of various vegetative organs. Live organism immunolabeling of cell wall polymers employing light microscopy and confocal laser scanning microscopy can be effectively performed using a large microplate-based screening strategy (see Figs. 1 and 2). Rapid cryofixation and imaging of variable pressure scanning electron microscopy also allows for imaging of surface features of all portions of the plant as clearly seen in Fig. 3.

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, as it has not been detected in unicellular algae. Here we present callose at the cytokinesis junction of the unicellular charophyte, P. 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 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.

Cutin and suberin are hydrophobic lipid biopolyester components of the cell walls of specialized plant tissue and cell-types, where they facilitate adaptation to terrestrial habitats. Many steps in their biosynthetic pathways have been characterized, but the basis of their spatial deposition and precursor trafficking is not well understood. Members of the GDSL lipase/esterase family catalyze cutin polymerization, and candidate proteins have been proposed to mediate interactions between cutin or suberin and other wall components. Comparative genomic studies of charophyte algae and early diverging land plants, combined with knowledge of the biosynthesis, trafficking and assembly mechanisms, suggests an origin for the capacity to secrete waxes, as well as aliphatic and phenolic compounds before the first colonization of true terrestrial habitats.

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., A layered endomembrane system consisting of linear rows of Golgi bodies and a peripheral vesicle network fuel the secretion of the complex extracellular matrix of the unicellular charophyte, Penium.

Background The 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. Results We 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. Conclusion Protoplast 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.

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.

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.

The adhesion of plant cells is vital for support and protection of the plant body and is maintained by a variety of molecular associations between cell wall components. In some specialized cases, though, plant cells are programmed to detach, and root cap-derived border cells are examples of this. Border cells (in some species known as border-like cells) provide an expendable barrier between roots and the environment. Their maturation and release is an important but poorly characterized cell separation event. To gain a deeper insight into the complex cellular dynamics underlying this process, we undertook a systematic, detailed analysis of pea (Pisum sativum) root tip cell walls. Our study included immunocarbohydrate microarray profiling, monosaccharide composition determination, Fourier-transformed infrared microspectroscopy, quantitative reverse transcription-PCR of cell wall biosynthetic genes, analysis of hydrolytic activities, transmission electron microscopy, and immunolocalization of cell wall components. Using this integrated glycobiology approach, we identified multiple novel modes of cell wall structural and compositional rearrangement during root cap growth and the release of border cells. Our findings provide a new level of detail about border cell maturation and enable us to develop a model of the separation process. We propose that loss of adhesion by the dissolution of homogalacturonan in the middle lamellae is augmented by an active biophysical process of cell curvature driven by the polarized distribution of xyloglucan and extensin epitopes.

International audience; Although plants and many algae (e.g. the Phaeophyceae, brown, and Rhodophyceae, red) are only very distantly related they are united in their possession of carbohydrate-rich cell walls, which are of integral importance being involved in many physiological processes. Furthermore,wall components have applications within food, fuel, pharmaceuticals, fibres (e.g. for textiles and paper) and building materials and have long been an active topic of research. As shown in the 27 papers in this Special Issue, as the major deposit of photosynthetically fixed carbon, and therefore energy investment, cell walls are of undisputed importance to the organisms that possess them, the photosynthetic eukaryotes ( plants and algae). The complexities of cell wall components along with their interactions with the biotic and abiotic environment are becoming increasingly revealed. The importance of plant and algal cell walls and their individual components to the function and survival of the organism, and for a number of industrial applications, are illustrated by the breadth of topics covered in this issue, which includes papers concentrating on various plants and algae, developmental stages, organs, cell wall components, and techniques. Although we acknowledge that there are many alternative ways in which the papers could be categorized (and many would fit within several topics), we have organized them as follows: (1) cell wall biosynthesis and remodelling, (2) cell wall diversity, and (3) application of new technologies to cell walls. Finally, we will consider future directions within plant cell wall research. Expansion of the industrial uses of cell walls and potentially novel uses of cell wall components are both avenues likely to direct future research activities. Fundamentally, it is the continued progression from characterization (structure, metabolism, properties and localization) of individual cell wall components through to defining their roles in almost every aspect of plant and algal physiology that will present many of the major challenges in future cell wall research.

Background and Aims: Root hairs are responsible for water and nutrient uptake from the soil and their growth is responsive to biotic and abiotic changes in their environment. Root hair expansion is a polarized process requiring secretory and endosomal pathways that deliver and recycle plasma membrane and cell wall material to the growing root hair tip. In this paper, the role of VTI13 (AT3G29100), a member of the VTI vesicular soluble NSF attachment receptor (SNARE) gene family in Arabidopsis thaliana, in root hair growth is described.\ud \ud Methods: Genetic analysis and complementation of the vti13 root hair phenotypes of Arabidopsis thaliana were first used to assess the role of VTI13 in root hair growth. Transgenic lines expressing a green fluorescent protein (GFP)–VTI13 construct were used to characterize the intracellular localization of VTI13 in root hairs using confocal microscopy and immunotransmission electron microscopy.\ud \ud Key Results: VTI13 was characterized and genetic analysis used to show that its function is required for root hair growth. Expression of a GFP–VTI13 fusion in the vti13 mutant background was shown to complement the vti13 root hair phenotype. GFP–VTI13 localized to both the vacuole membrane and a mobile endosomal compartment. The function of VTI13 was also required for the localization of SYP41 to the trans-Golgi network. Immunohistochemical analysis indicated that cell wall organization is altered in vti13 root hairs and root epidermal cells.\ud \ud Conclusions: These results show that VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole within root hairs and is essential for the maintenance of cell wall organization and root hair growth in arabidopsis.

Summary Pectic homogalacturonan (HG) is one of the main constituents of plant cell walls. When processed to low degrees of esterification, HG can form complexes with divalent calcium ions. These macromolecular structures (also called egg boxes) play an important role in determining the biomechanics of cell walls and in mediating cell-to-cell adhesion. Current immunological methods enable only steady-state detection of egg box formation in situ. Here we present a tool for efficient real-time visualisation of available sites for HG crosslinking within cell wall microdomains. Our approach is based on calcium-mediated binding of fluorescently tagged long oligogalacturonides (OGs) with endogenous de-esterified HG. We established that more than seven galacturonic acid residues in the HG chain are required to form a stable complex with endogenous HG through calcium complexation in situ, confirming a recently suggested thermodynamic model. Using defined carbohydrate microarrays, we show that the long OG probe binds exclusively to HG that has a very low degree of esterification and in the presence of divalent ions. We used this probe to study real-time dynamics of HG during elongation of Arabidopsis pollen tubes and root hairs. Our results suggest a different spatial organisation of incorporation and processing of HG in the cell walls of these two tip-growing structures.

Algae constitute a large and diverse array of photosynthetic eukaryotes that are common to most of modern earth’s photic zones. Algae are critical to global primary production, CO2 sequestering and biomineralization as well as being of economic significance in the food, pharmaceutical and biofuel industries. The extracellular matrix of algal cells, most notably the cell wall, is of fundamental significance to survival and often serves as the major product of photosynthetic carbon fixation. The cell walls of most algae consist of a framework of fibrillar polysaccharides that are embedded in a matrix composed of neutral and charged polysaccharides along with various proteins, phenolics and complexed cations. The fibrillar components include mannans, xylans and most notably, cellulose, whose synthesis occurs in membrane-bound enzyme complexes. These load-bearing fibrillar components are inserted in complex networks of polysaccharides that include hemicelluloses and polyanionic polymers such as pectins, alginates, fucoidans and the sulfated galactans of red algae, agar and carageenan. These polymers are synthesized in the Golgi Apparatus, transported to cell surface sites via actin- and tubulin-based motors and deposited in the wall complex. Often, post-secretory crosslinking with cations alters the structural architecture of these polymers that, in turn, influences the strength and function of the cell wall. Proteins are also found in algal cell walls including highly glycosylated and hydroxyproline-rich forms, some of which have structural semblance to extensins and arabinogalactan proteins of land plants. Modern molecular and immunobinding studies are now probing the specific mechanisms in wall development including modulations that occur during morphogenesis and in response to environmental triggers.

Penium margaritaceumis a unicellular charophycean green alga that possesses cell wall polymers similar to those of land plants. Several wall macromolecules of this alga are recognized by monoclonal antibodies specific for wall polymer epitopes of land plants. Immunofluorescence protocols using these antibodies may be employed to label specific cell wall constituents of live cells. Fluorescent labeling persists for several days, and this attribute allows for tracing of wall epitopes in both long- and short-term studies of cell development. Quantitative analysis of surface area covered by cell wall polymers is also easily performed. We show that significant cell expansion caused by incubation of cells in low levels of osmotically active agents like mannitol, glucose, or sucrose results from the inability of cells to undergo cytokinesis but does not result in significant changes to the amount of new cell wall. We also demonstrate that cells can be maintained for long periods of time in culture medium supplemented with specific cell wall-degrading enzymes where notable changes to wall infrastructure occur. These results demonstrate the great potential value ofPeniumin elucidating fundamental events during cell wall synthesis and modulation in plant cells.

Immunocytochemistry, comprehensive microarray polymer profiling (CoMPP), and biochemical analysis were used to elucidate the distribution of polymers in the cell walls of seven distinct parts of the shoot region of the thallus of the charophycean green alga Chara corallina. The cell walls contain a diverse set of polymers, many of which are commonly found in the cell walls of land plants, including cellulose, pectic polymers, arabinogalactan proteins, and several polymers that are classified as cross‐linking polysaccharides in embryophytes, including heteroxylans and heteromannans. Evidence of the presence of xyloglucan was also found. Cellulose and homogalacturonan were found in virtually all parts of the vegetative and reproductive shoot thallus, whereas the occurrence of other polymers in the cell walls within the different cell types was more varied. These results, which demonstrate the shared biochemical characteristics of the cell walls of Chara and of embryophytes, support previous molecular, ultra...

Plant cell walls are highly sophisticated fiber composite structures that have evolved to fulfill a wide range of biological roles that are central to plant life. In so doing they have diversified not just between species, but also within plants, between cell types, and between cell wall

†Background and Aims The production of multicellular gametangia in green plants represents an early evolutionary development that is found today in all land plants and advanced clades of the Charophycean green algae. The processing of cell walls is an integral part of this morphogenesis yet very little is known about cell wall dynamics in early-divergent green plants such as the Charophycean green algae. This study represents a comprehensive analysis of antheridium development and spermatogenesis in the green alga, Chara corallina. †Methods Microarrays of cell wall components and immunocytochemical methods were employed in order to analyse cell wall macromolecules during antheridium development. †Key Results Cellulose and pectic homogalacturonan epitopes were detected throughout all cell types of the developing antheridium including the unique cell wall protuberances of the shield cells and the cell walls of sperm cell initials. Arabinogalactan protein epitopes were distributed only in the epidermal shield cell layers and anti-xyloglucan antibody binding was only observed in the capitulum region that initially yields the sperm filaments. During the terminal stage of sperm development, no cell wall polymers recognized by the probes employed were found on the scale-covered sperm cells. †Conclusions Antheridium development in C. corallina is a rapid event that includes the production of cell walls that contain polymers similar to those found in land plants. While pectic and cellulosic epitopes are ubiquitous in the antheridium, the distribution of arabinogalactan protein and xyloglucan epitopes is restricted to specific zones. Spermatogenesis also includes a major switch in the production of extracellular matrix macromolecules from cell walls to scales, the latter being a primitive extracellular matrix characteristic of green plants.

Cell-wall (CW) development in the desmid Penium margaritaceum (Ehrenb.) Breb. was studied using immunofluorescence labeling of living cells with the monoclonal antibodies (mAbs) JIM5 and JIM7, which recognize unesterified and methyl-esterified homogalacturonan (HG), respectively. During cell expansion, HG was secreted in a high-esterified form at a narrow band, called the HG secretion band or HGSB, at the isthmus or the polar tip of a daughter semicell. As newly secreted HG is displaced outward on the cell surface, deesterification and subsequent calcium (Ca(2+) )-complexing occurred to yield a rigid covering. HG secretion and CW/cell expansion were reversibly inhibited by dark, brefeldin A (BFA), and incubation in 0.24-0.36 M sucrose but were not altered by treatment with actin/microfilament drugs. The HGSB was detected near the nucleus during most cell-cycle events. Centrifugation displaced the nucleus away from the HGSB, but HG synthesis was not affected. HGSB activity was preceded by, and coordinated with, Calcofluor labeling, which suggests that cellulose production in CW/cell-expansion sites was critical to expansion control. In many first-cell-division products, asymmetric patterning of HG was noted in the CW. These asymmetric patterns most likely were a result of timing mechanisms and displacement of the nucleus-HGSB during the cell cycle.

Cosmarium reniforme (Zygnematophyceae, Streptophyta) is a green alga that is commonly found in biofilms of wetlands of the Adirondack region, NY (USA). Two distinctive characteristics that are critical to this alga's survival in a benthic biofilm are its elaborate cell morphology and extracellular matrix (ECM). In this study, ultrastructural, immunocytochemical, and experimental methodologies were employed in order to elucidate the cellular characteristics that are critical for survival in a biofilm. The ECM consists of a thick, outwardly lobed cell wall (CW), which contains a patterned network of structurally complex pores. Each pore consists of a narrow channel, terminating internally at a bulb that invaginates localized regions of the plasma membrane. The outer region of the pore contains arabinogalactan protein-like and extensin epitopes that are likely involved in adhesion mechanisms of the cell. External to the CW is the extracellular polymeric substance that is employed in ensheathment of the cell to the substrate and in gliding motility. The architectural design/biochemical make-up of the CW and a secretory system that encompasses the coordinated activities of the endomembrane and cytomotile/cytoskeletal systems provide the organism with effective mechanisms to support life within the biofilm complex.

Pleurotaenium trabecula (Ehren.) Nageli is a placoderm desmid that commonly occurs in wetland biofilms of the southeastern Adirondacks (NY, USA). It often displays a distinctive habit whereby the cell remains attached to the substrate via the polar end of one semicell, while the remainder of the cell is suspended in the water column. In this study, we examined the extracellular matrix (ECM) of this alga to elucidate its adhesion mechanisms and postadhesion behavior. The ECM consists of the following: (i) an extracellular polymeric substance (EPS), which includes polyanionic and sulfated polysaccharides; (ii) a thin pectin-containing primary cell wall, which is quickly sloughed off after postcytokinetic semicell expansion; and (iii) a thick secondary cell wall that is perforated with a distinct pore complex. Each pore of this complex possesses an external network of densely aggregated fibrils. Selective solubilization and immunolabeling studies suggest that these fibrillar aggregates or “adhesion centers” (i.e., ACs) contain arabinogalactan protein and are involved in initial adhesion of the cell to a substrate. We propose that postinitial adhesion behavior entails localized secretion of EPS derived from a large pool of EPS-containing vesicles situated in the peripheral cytoplasm. As the EPS absorbs water, hygroscopic pressure breaks the connections between the ACs on the cell wall and substrate and allows a portion of a cell to lift up into the water column.

Plant cell walls are essential for proper growth, development, and interaction with the environment. It is generally accepted that land plants arose from aquatic ancestors which are sister groups to the charophycean algae (i.e., Streptophyta), and study of wall evolution during this transition promises insight into structure-function relationships of wall components. In this paper, we explore wall evolutionary history by studying the incorporation of pectin polymers into cell walls of the model organism Penium margaritaceum, a simple single-cell desmid. This organism produces only a primary wall consisting of three fibrillar or fibrous layers, with the outermost stratum terminating in distinct, calcified projections. Extraction of isolated cell walls with trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid yielded a homogalacturonan (HGA) that was partially methyl esterified and equivalent to that found in land plants. Other pectins common to land plants were not detected, although selected components of some of these polymers were present. Labeling with specific monoclonal antibodies raised against higher-plant HGA epitopes (e.g., JIM5, JIM7, LM7, 2F4, and PAM1) demonstrated that the wall complex and outer layer projections were composed of the HGA which was significantly calcium complexed. JIM5 and JIM7 labeling suggested that highly methyl esterified HGA was secreted into the isthmus zone of dividing cells, the site of active wall secretion. As the HGA was displaced to more polar regions, de-esterification in a non-blockwise fashion occurred. This, in turn, allowed for calcium binding and the formation of the rigid outer wall layer. The patterning of HGA deposition provides interesting insights into the complex process of pectin involvement in the development of the plant cell wall.

Closterium acerosum Ehrenberg (Chlorophyta) produced a distinct network of thin cytoplasmic strands, or Hechtian strands, upon controlled plasmolysis in a sucrose solution. The strands persisted for 30 min or longer and could be visualized with both LM and EM. Near the plasma membrane of the polar zones of plasmolyzing protoplasts, the strands formed a "lattice"-like arrangement with interstrand spacing of 120-130 nm. The strands terminated at the fibrous zone of the inner cell wall stratum. Although actin cables could be found attached to the plasma membrane upon rhodamine phalloidin labeling of membrane ghosts, neither microfilaments nor microtubules were found in Hechtian strands at any stage of development. The formation of strands was not disrupted by centrifugation at 8000 g or by repeated cycles of plasmolysis-deplasmolysis. Application of microtubule- or microfilament-affecting agents or various proteolytic/polysaccharide-degrading enzymes did not disrupt the formation of strands. Cold treatment of cells resulted in the formation of Hechtian strands.

The Charophycean green algae (CGA) occupy a key phylogenetic position as the evolutionary grade that includes the sister group of the land plants (embryophytes), and so provide potentially valuable experimental systems to study the development and evolution of traits that were necessary for terrestrial colonization. The nature and molecular bases of such traits are still being determined, but one critical adaptation is thought to have been the evolution of a complex cell wall. Very little is known about the identity, origins and diversity of the biosynthetic machinery producing the major suites of structural polymers (i. e., cell wall polysaccharides and associated molecules) that must have been in place for land colonization. However, it has been suggested that the success of the earliest land plants was partly based on the frequency of gene duplication, and possibly whole genome duplications, during times of radical habitat changes. Orders of the CGA span early diverging taxa retaining more ancestral characters, through complex multicellular organisms with morphological characteristics resembling those of land plants. Examination of gene diversity and evolution within the CGA could help reveal when and how the molecular pathways required for synthesis of key structural polymers in land plants arose.

The growth of a plant cell encompasses a complex set of subcellular components interacting in a highly coordinated fashion. Ultimately, these activities create specific cell wall structural domains that regulate the prime force of expansion, internally generated turgor pressure. The precise organization of the polymeric networks of the cell wall around the protoplast also contributes to the direction of growth, the shape of the cell, and the proper positioning of the cell in a tissue. In essence, plant cell expansion represents the foundation of development. Most studies of plant cell expansion have focused primarily upon late divergent multicellular land plants and specialized cell types (e.g., pollen tubes, root hairs). Here, we describe a unicellular green alga, Penium margaritaceum (Penium), which can serve as a valuable model organism for understanding cell expansion and the underlying mechanics of the cell wall in a single plant cell.

The cell wall is directly involved in cell growth, and its ability to loosen and rearrange allows for cell expansion through the existing turgor pressure. Thus, information on cell wall deposition and rearrangement can provide insights into the overall plant growth. This chapter describes two methods that can be used to evaluate cell expansion (1) in the model plant Arabidopsis thaliana and (2) the model alga Penium margaritaceum. These methods are further used to screen for small molecules that induce cell growth phenotypic changes affecting cell wall. Identification of such small molecules is beneficial due to their posttranslational mechanism of action that can be controlled in a temporal and spatial manner. Chemical genomics has the ability to overcome issues of genetic redundancy and lethality, which can hinder traditional genetic methods. The identification of small molecules in these screens will provide useful information on plant cell wall biology and overall plant growth.

The growth of a plant cell encompasses a complex set of subcellular components interacting in a highly coordinated fashion. Ultimately, these activities create specific cell wall structural domains that regulate the prime force of expansion, internally generated turgor pressure. The precise organization of the polymeric networks of the cell wall around the protoplast also contributes to the direction of growth, the shape of the cell, and the proper positioning of the cell in a tissue. In essence, plant cell expansion represents the foundation of development. Most studies of plant cell expansion have focused primarily upon late divergent multicellular land plants and specialized cell types (e.g., pollen tubes, root hairs). Here, we describe a unicellular green alga, Penium margaritaceum (Penium), which can serve as a valuable model organism for understanding cell expansion and the underlying mechanics of the cell wall in a single plant cell.

Background and Aims Parasitic plants obtain nutrients from their hosts through organs called haustoria. The hyaline body is a specialized parenchymatous tissue occupying the central parts of haustoria in many Orobanchaceae species. The structure and functions of hyaline bodies are poorly understood despite their apparent necessity for the proper functioning of haustoria. Reported here is a cell wall-focused immunohistochemical study of the hyaline bodies of three species from the ecologically important clade of rhinanthoid Orobanchaceae. Methods Haustoria collected from laboratory-grown and field-collected plants of Rhinanthus minor, Odontites vernus and Melampyrum pratense attached to various hosts were immunolabelled for cell wall matrix glycans and glycoproteins using specific monoclonal antibodies (mAbs). Key Results Hyaline body cell wall architecture differed from that of the surrounding parenchyma in all species investigated. Enrichment in arabinogalactan protein (AGP) epitopes labelled with mAbs LM2, JIM8, JIM13, JIM14 and CCRC-M7 was prominent and coincided with reduced labelling of de-esterified homogalacturonan with mAbs JIM5, LM18 and LM19. Furthermore, paramural bodies, intercellular deposits and globular ergastic bodies composed of pectins, xyloglucans, extensins and AGPs were common. In Rhinanthus they were particularly abundant in pairings with legume hosts. Hyaline body cells were not in direct contact with haustorial xylem, which was surrounded by a single layer of paratracheal parenchyma with thickened cell walls abutting the xylem. Conclusions The distinctive anatomy and cell wall architecture indicate hyaline body specialization. Altered proportions of AGPs and pectins may affect the mechanical properties of hyaline body cell walls. This and the association with a transfer-like type of paratracheal parenchyma suggest a role in nutrient translocation. Organelle-rich protoplasts and the presence of exceptionally profuse intra-and intercellular wall materials when attached to a nitrogen-fixing host suggest subsequent processing and transient storage of nutrients. AGPs might therefore be implicated in nutrient transfer and metabolism in haustoria.

The pectin polymer homogalacturonan (HG) is a major component of land plant cell walls and is especially abundant in the middle lamella. Current models suggest that HG is deposited into the wall as a highly methylesterified polymer, demethylesterified by pectin methylesterase enzymes and cross-linked by calcium ions to form a gel. However, this idea is based largely on indirect evidence and in vitro studies. We took advantage of the wall architecture of the unicellular alga Penium margaritaceum, which forms an elaborate calcium cross-linked HG-rich lattice on its cell surface, to test this model and other aspects of pectin dynamics. Studies of live cells and microscopic imaging of wall domains confirmed that the degree of methylesterification and sufficient levels of calcium are critical for lattice formation in vivo. Pectinase treatments of live cells and immunological studies suggested the presence of another class of pectin polymer, rhamnogalacturonan I, and indicated its colocalization and structural association with HG. Carbohydrate microarray analysis of the walls of P. margaritaceum, Physcomitrella patens, and Arabidopsis (Arabidopsis thaliana) further suggested the conservation of pectin organization and interpolymer associations in the walls of green plants. The individual constituent HG polymers also have a similar size and branched structure to those of embryophytes. The HG-rich lattice of P. margaritaceum, a member of the charophyte green algae, the immediate ancestors of land plants, was shown to be important for cell adhesion. Therefore, the calcium-HG gel at the cell surface may represent an early evolutionary innovation that paved the way for an adhesive middle lamella in multicellular land plants. peer-reviewed

Background and aims Penium margaritaceum is a unicellular charophycean green alga with a unique bi-directional polar expansion mechanism that occurs at the central isthmus zone prior to cell division. This entails the focused deposition of cell-wall polymers coordinated by the activities of components of the endomembrane system and cytoskeletal networks. The goal of this study was to elucidate the structural organization of the cortical cytoskeletal network during the cell cycle and identify its specific functional roles during key cell-wall developmental events: pre-division expansion and cell division. Methods Microtubules and actin filaments were labelled during various cell cycle phases with an anti-tubulin antibody and rhodamine phalloidin, respectively. Chemically induced disruption of the cytoskeleton was used to elucidate specific functional roles of microtubules and actin during cell expansion and division. Correlation of cytoskeletal dynamics with cell-wall development included live cell labelling with wall polymer-specific antibodies and electron microscopy. Key results The cortical cytoplasm of Penium is highlighted by a band of microtubules found at the cell isthmus, i.e. the site of pre-division wall expansion. This band, along with an associated, transient band of actin filaments, probably acts to direct the deposition of new wall material and to mark the plane of the future cell division. Two additional bands of microtubules, which we identify as satellite bands, arise from the isthmus microtubular band at the onset of expansion and displace toward the poles during expansion, ultimately marking the isthmus of future daughter cells. Treatment with microtubule and actin perturbation agents reversibly stops cell division. Conclusions The cortical cytoplasm of Penium contains distinct bands of microtubules and actin filaments that persist through the cell cycle. One of these bands, termed the isthmus microtubule band, or IMB, marks the site of both pre-division wall expansion and the zone where a cross wall will form during cytokinesis. This suggests that prior to the evolution of land plants, a dynamic, cortical cytoskeletal array similar to a pre-prophase band had evolved in the charophytes. However, an interesting variation on the cortical band theme is present in Penium, where two satellite microtubule bands are produced at the onset of cell expansion, each of which is destined to become an IMB in the two daughter cells after cytokinesis. These unique cytoskeletal components demonstrate the close temporal control and highly coordinated cytoskeletal dynamics of cellular development in Penium.

Polysaccharides are major components of extracellular matrices and are often extensively modified post-synthetically to suit local requirements and developmental programmes. However, our current understanding of the spatiotemporal dynamics and functional significance of these modifications is limited by a lack of suitable molecular tools. Here, we report the development of a novel non-immunological approach for producing highly selective reciprocal oligosaccharide-based probes for chitosan (the product of chitin deacetylation) and for demethylesterified homogalacturonan. Specific reciprocal binding is mediated by the unique stereochemical arrangement of oppositely charged amino and carboxy groups. Conjugation of oligosaccharides to fluorophores or gold nanoparticles enables direct and rapid imaging of homogalacturonan and chitosan with unprecedented precision in diverse plant, fungal and animal systems. We demonstrated their potential for providing new biological insights by using them to study homogalacturonan processing during Arabidopsis thaliana root cap development and by analyzing sites of chitosan deposition in fungal cell walls and arthropod exoskeletons.

Closterium acerosum Ehrenberg (Chlorophyta) possesses a trilayered cell wall consisting of an outer tri-laminate stratum, a fibrous middle layer, and a thick inner fibrous layer. The outermost layer has a series of external parallel ridges and valleys. At the bases of the valleys are the wall pores, the site of mucilage release. Pure fractions of cell walls were isolated and inclusive pectin and wall protein fractions were extracted and characterized. Two pectin-like fractions were isolated: a CDTA-extracted polymer consisting of 60.1% galacturonic acid and a Na2CO3-extracted fraction consisting of 39.9% galacturonic acid. Two major protein fractions, one with a molecular mass of 23.5 kDa and one with a molecular mass of 28.5 kDa, were isolated by preparative gel electrophoresis. The former was glycine-rich, whereas the latter contained both significant amounts of glycine and hydroxyproline. Antibodies were raised to both the pectin fractions and the 23.5-kDa wall protein fraction. Immunocytochemical labeling of whole cells and wall fragments using antibodies raised against CDTA and Na2CO3 extracts showed that these pectin-like components were found throughout the wall strata and were more concentrated at the polar tips, the site of new wall synthesis in growing semicells. Immunogold labeling showed that their production was focused on the trans-Golgi network of the Golgi apparatus. Immunolabeling with an antibody raised against the 23.5-kDa glycine-rich wall protein showed close association of the protein with the wall pores. Similarly, immunogold labeling revealed that the protein was processed throughout the entire Golgi body even when large mucilage-containing vesicles were being processed. The roles of the secretory apparatus and putative spitzenkorper-like regions of the cell are discussed.

Closterium acerosum is a large bipolar desmid that has an extensive secretory apparatus. Two major secretory complexes that are produced by this green alga are a glucuronic acid–rich mucilage that is key in the gliding mechanism and a cell wall consisting of multiple polysaccharide and protein components. These substances are synthesized and processed for secretion through an elaborate endomembrane system and are transported around the cell and to the cell surface via actin‐mediated cyclosis. In this study, an analysis of the role of calmodulin with regard to the secretory apparatus was conducted. Whole‐cell extracts and Western blot analysis show the existence of calmodulin. Immunogold labeling reveals calmodulin in the peripheral cytoplasm near the plasmamembrane and cell wall region. Calmodulin antagonists cause significant disruption to the morphology of the Golgi apparatus. On application of the antagonists W7, W13, TMB‐8, and TFP, the Golgi body exhibits reduction in the number of cisternae per stac...

Application of the dintroaniline compound, oryzalin, which inhibits microtubule formation, to the unicellular green alga Penium margaritaceum caused major perturbations to its cell morphology, such as swelling at the wall expansion zone in the central isthmus region. Cell wall structure was also notably altered, including a thinning of the inner cellulosic wall layer and a major disruption of the homogalacturonan (HG)-rich outer wall layer lattice. Polysaccharide microarray analysis indicated that the oryzalin treatment resulted in an increase in HG abundance in treated cells but a decrease in other cell wall components, specifically the pectin rhamnogalacturonan I (RG-I) and arabinogalactan proteins (AGPs). The ring of microtubules that characterizes the cortical area of the cell isthmus zone was significantly disrupted by oryzalin, as was the extensive peripheral network of actin microfilaments. It is proposed that the disruption of the microtubule network altered cellulose production, the main load-bearing component of the cell wall, which in turn affected the incorporation of HG in the two outer wall layers, suggesting coordinated mechanisms of wall polymer deposition.

The charophyte green algae (CGA, Streptophyta, Viridiplantae) occupy a key phylogenetic position as the immediate ancestors of land plants but, paradoxically, are less well-studied than the other major plant lineages. This is particularly true in the context of functional genomic studies, where the lack of an efficient protocol for their stable genetic transformation has been a major obstacle. Observations of extant CGA species suggest the existence of some of the evolutionary adaptations that had to occur for land colonization; however, to date, there has been no robust experimental platform to address this genetically. We present a protocol for high-throughput Agrobacterium tumefaciens-mediated transformation of Penium margaritaceum, a unicellular CGA species. The versatility of Penium as a model for studying various aspects of plant cell biology and development was illustrated through non-invasive visualization of protein localization and dynamics in living cells. In addition, the utility of RNA interference (RNAi) for reverse genetic studies was demonstrated by targeting genes associated with cell wall modification (pectin methylesterase) and biosynthesis (cellulose synthase). This provided evidence supporting current models of cell wall assembly and inter-polymer interactions that were based on studies of land plants, but in this case using direct observation in vivo. This new functional genomics platform has broad potential applications, including studies of plant organismal biology and the evolutionary innovations required for transition from aquatic to terrestrial habitats.

The green algae represent a large group of morphologically diverse photosynthetic eukaryotes that occupy virtually every photic habitat on the planet. The extracellular coverings of green algae including cell walls are also diverse. A recent surge of research in green algal cell walls fueled by new emerging technologies has revealed new and critical insight concerning these coverings. For example, the late divergent taxa of the Charophycean green algae possess cell walls containing assemblages of polymers with notable similarity to the cellulose, pectins, hemicelluloses, arabinogalactan proteins (AGPs), extensin, and lignin present in embryophyte walls. Ulvophycean seaweeds have cell wall components whose most abundant fibrillar constituents may change from cellulose to β-mannans to β-xylans and during different life cycle phases. Likewise, these algae produce complex sulfated polysaccharides, AGPs, and extensin. Chlorophycean green algae produce a wide array of walls ranging from cellulose-pectin complexes to ones made of hydroxyproline-rich glycoproteins. Larger and more detailed surveys of the green algal taxa including incorporation of emerging genomic and transcriptomic data are required in order to more fully resolve evolutionary trends within the green algae and in relationship with higher plants as well as potential applications of wall components in the food and pharmaceutical industries. Fil: Domozych, David S.. Skidmore College; Estados Unidos Fil: Ciancia, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Centro de Investigaciones en Hidratos de Carbono. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigaciones en Hidratos de Carbono; Argentina Fil: Fangel, Jonatan U.. University Of Copenhagen, Faculty Of Life Sciences; Dinamarca Fil: Mikkelsen, Maria Dalgaard. Universidad de Copenhagen; Dinamarca Fil: Ulvskov, Peter. Universidad de Copenhagen; Dinamarca Fil: Willats, William G. T.. Universidad de Copenhagen; Dinamarca

A morphological analysis of the plasma membrane and peripheral endomembrane components of the unicellular chlamydomonad flagellate, Gloeomonas kupfferi, was performed. Conventional fixation, freeze substitution, and rapid freeze-deep etch processing protocols for electron microscopic analyses revealed the following. The plasma membrane is highlighted by distinct infoldings which do not appear to be sensitive to changes in osmotic or cell cycle conditions. These infoldings are irregularly-spaced and roughly 200-225 nm apart. Each elliptical infolding is 400-440 nm long and 90-115 nm wide. The exoplasmic face (EF) of each infolding is highlighted by an aggregation of 130-160 nm intramembranous particles (imps) that are 9.1-10·5 nm in size. These infoldings are associated with the peripheral endoplasmic reticulum network and microtubular network internally and the inner wall layer externally. It is suggested that these infoldings may be associated with cell wall maintenance.

Algal cell walls are composed of a diverse array of fibrillar, matrix and crystalline polymers interacting with various ions and water. The diverse array of cell walls exhibited in the various algal groups is a manifestation of ancient evolutionary origins and ecological pressures of modern earth habitats. Cell walls often represent the dominant component of the extracellular matrix and represent the largest or a significant percentage of the photosynthetically fixed carbon of the algae. Walls are typically fibrous composites of microfibrillar polysaccharides embedded in matrix polysaccharides and proteoglycans. Scales and extracellular polymeric substances may also constitute the algal extracellular matrix. The production of the cell walls requires the highly coordinated interaction of several subcellular systems and is controlled by complex gene expression programmes. Modern technologies employed from immunology, molecular genetics, biochemistry and microscopy-based imaging are transforming our understanding of the cell structure, functions and development. Key Concepts: Algae represent a diverse group of photosynthetic eukaryotes with a wide range of cell wall types. New technologies derived from immunology, microscopy-based imaging, molecular genetics and biochemistry are greatly enhancing our understanding of algal cell walls. Primitive green algae of the Prasinophyceae produce layers of multishaped scales. The Charophycean green algae produce cell walls containing polymers similar to land plants. The red algae possess complex composite cell walls made of cellulose, xylan or mannan fibrils and extensive matrix polysaccharides including the economically important carrageenan and agar. Diatoms produce highly sculpted frustules made of silica-based composites and often produce extensive stalks and other extracellular polymeric substances. Brown algae produce cell walls containing cellulose, matrix polysaccharides and in some cases, phenolics. Keywords: cellulose; pectins; scales; frustule; carageenan; align; morphogenesis; algae