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 extracellular matrix of green algae.

Green algae display a wide range of extracellular matrix (ECM) components that include various types of cell walls (CW), scales, crystalline glycoprotein coverings, hydrophobic compounds, and complex gels or mucilage. Recently, new information derived from genomic/transcriptomic screening, advanced biochemical analyses, immunocytochemical studies, and ecophysiology has significantly enhanced and refined our understanding of the green algal ECM. In the later diverging charophyte group of green algae, the CW and other ECM components provide insight into the evolution of plants and the ways the ECM modulates during environmental stress. Chlorophytes produce diverse ECM components, many of which have been exploited for various uses in medicine, food, and biofuel production. This review highlights major advances in ECM studies of green algae.

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

BACKGROUND AND AIMS: Endosidins are a group of low-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 the effects of endosidin 5 (ES5) on the Golgi apparatus and the secretion of extracellular matrix (ECM) components in 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 ES5. METHODS: 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 to detail the changes to the Golgi apparatus. KEY RESULTS: While other endosidins were able to impact EPS secretion and cell wall expansion, only ES5 completely inhibited EPS secretion and cell wall expansion over 24 h. 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. CONCLUSIONS: ES5 alters secretion of ECM material in Penium by affecting the Golgi apparatus and does so in a markedly different way from other endomembrane inhibitors such as brefeldin A and concanamycin A.

The cell biology of charophytes: Exploring the past and models for the future.

Charophytes (Streptophyta) represent a diverse assemblage of extant green algae that are the sister lineage to land plants. About 500-600+ million years ago, a charophyte progenitor successfully colonized land and subsequently gave rise to land plants. Charophytes have diverse but relatively simple body plans that make them highly attractive organisms for many areas of biological research. At the cellular level, many charophytes have been used for deciphering cytoskeletal networks and their dynamics, membrane trafficking, extracellular matrix secretion, and cell division mechanisms. Some charophytes live in challenging habitats and have become excellent models for elucidating the cellular and molecular effects of various abiotic stressors on plant cells. Recent sequencing of several charophyte genomes has also opened doors for the dissection of biosynthetic and signaling pathways. While we are only in an infancy stage of elucidating the cell biology of charophytes, the future application of novel analytical methodologies in charophyte studies that include a broader survey of inclusive taxa will enhance our understanding of plant evolution and cell dynamics.

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.

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 ß-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.

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.

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.

Rapid Assessment of Cell Wall Polymer Distribution and Surface Topology of Arabidopsis Seedlings.

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 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.

Cutin and suberin: assembly and origins of specialized lipidic cell wall scaffolds.

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.

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.

Arabinogalactan Proteins and the Extracellular Matrix of Charophytes: A Sticky Business.

Charophytes represent the group of green algae whose ancestors invaded land and ultimately gave rise to land plants 450 million years ago. While Zygnematophyceae are believed to be the direct sister lineage to embryophytes, different members of this group (Penium, Spirogyra, Zygnema) and the advanced thallus forming Coleochaete as well as the sarcinoid basal streptophyte Chlorokybus were investigated concerning their vegetative extracellular matrix (ECM) properties. Many taxa exhibit adhesion phenomena that are critical for affixing to a substrate or keeping cells together in a thallus, however, there is a great variety in possible reactions to e.g., wounding. In this study an analysis of adhesion mechanisms revealed that arabinogalactan proteins (AGPs) are most likely key adhesion molecules. Through use of monoclonal antibodies (JIM13) or the Yariv reagent, AGPs were located in cell surface sheaths and cell walls that were parts of the adhesion focal zones on substrates including wound induced rhizoid formation. JIM5, detecting highly methyl-esterfied homoglacturonan and JIM8, an antibody detecting AGP glycan and LM6 detecting arabinans were also tested and a colocalization was found in several examples (e.g., Zygnema) suggesting an interplay between these components. AGPs have been described in this study to perform both, cell to cell adhesion in algae forming thalli and cell to surface adhesion in the filamentous forms. These findings enable a broader evolutionary understanding of the function of AGPs in charophyte green algae.

A Hybrid Approach Enabling Large-Scale Glycomic Analysis of Post-Golgi Vesicles Reveals a Transport Route for Polysaccharides.

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 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.

Vacuolar Protein Sorting 26C encodes an evolutionarily conserved large retromer subunit in eukaryotes that is important for root hair growth in Arabidopsis thaliana.

The large retromer complex participates in diverse endosomal trafficking pathways and is essential for plant developmental programs, including cell polarity, programmed cell death and shoot gravitropism in Arabidopsis. Here we demonstrate that an evolutionarily conserved VPS26 protein (VPS26C; At1G48550) functions in a complex with VPS35A and VPS29 necessary for root hair growth in Arabidopsis. Bimolecular fluorescence complementation showed that VPS26C forms a complex with VPS35A in the presence of VPS29, and this is supported by genetic studies showing that vps29 and vps35a mutants exhibit altered root hair growth. Genetic analysis also demonstrated an interaction between a VPS26C trafficking pathway and one involving the SNARE VTI13. Phylogenetic analysis indicates that VPS26C, with the notable exception of grasses, has been maintained in the genomes of most major plant clades since its evolution at the base of eukaryotes. To test the model that VPS26C orthologs in animal and plant species share a conserved function, we generated transgenic lines expressing GFP fused with the VPS26C human ortholog (HsDSCR3) in a vps26c background. These studies illustrate that GFP-HsDSCR3 is able to complement the vps26c root hair phenotype in Arabidopsis, indicating a deep conservation of cellular function for this large retromer subunit across plant and animal kingdoms.

High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening.

Tomato (Solanum lycopersicum) is an established model for studying fruit biology; however, most studies of tomato fruit growth and ripening are based on homogenized pericarp, and do not consider the internal tissues, or the expression signatures of individual cell and tissue types. We present a spatiotemporally resolved transcriptome analysis of tomato fruit ontogeny, using laser microdissection (LM) or hand dissection coupled with RNA-Seq analysis. Regulatory and structural gene networks, including families of transcription factors and hormone synthesis and signaling pathways, are defined across tissue and developmental spectra. The ripening program is revealed as comprising gradients of gene expression, initiating in internal tissues then radiating outward, and basipetally along a latitudinal axis. We also identify spatial variations in the patterns of epigenetic control superimposed on ripening gradients. Functional studies elucidate previously masked regulatory phenomena and relationships, including those associated with fruit quality traits, such as texture, color, aroma, and metabolite profiles.

Development of novel monoclonal antibodies against starch and ulvan - implications for antibody production against polysaccharides with limited immunogenicity.

Monoclonal antibodies (mAbs) are widely used and powerful research tools, but the generation of mAbs against glycan epitopes is generally more problematic than against proteins. This is especially significant for research on polysaccharide-rich land plants and algae (Viridiplantae). Most antibody production is based on using single antigens, however, there are significant gaps in the current repertoire of mAbs against some glycan targets with low immunogenicity. We approached mAb production in a different way and immunised with a complex mixture of polysaccharides. The multiplexed screening capability of carbohydrate microarrays was then exploited to deconvolute the specificities of individual mAbs. Using this strategy, we generated a set of novel mAbs, including one against starch (INCh1) and one against ulvan (INCh2). These polysaccharides are important storage and structural polymers respectively, but both are generally considered as having limited immunogenicity. INCh1 and INCh2 therefore represent important new molecular probes for Viridiplantae research. Moreover, since the α-(1-4)-glucan epitope recognised by INCh1 is also a component of glycogen, this mAb can also be used in mammalian systems. We describe the detailed characterisation of INCh1 and INCh2, and discuss the potential of a non-directed mass-screening approach for mAb production against some glycan targets.

Cuticle Biosynthesis in Tomato Leaves Is Developmentally Regulated by Abscisic Acid.

The expansion of aerial organs in plants is coupled with the synthesis and deposition of a hydrophobic cuticle, composed of cutin and waxes, which is critically important in limiting water loss. While the abiotic stress-related hormone abscisic acid (ABA) is known to up-regulate wax accumulation in response to drought, the hormonal regulation of cuticle biosynthesis during organ ontogeny is poorly understood. To address the hypothesis that ABA also mediates cuticle formation during organ development, we assessed the effect of ABA deficiency on cuticle formation in three ABA biosynthesis-impaired tomato mutants. The mutant leaf cuticles were thinner, had structural abnormalities, and had a substantial reduction in levels of cutin. ABA deficiency also consistently resulted in differences in the composition of leaf cutin and cuticular waxes. Exogenous application of ABA partially rescued these phenotypes, confirming that they were a consequence of reduced ABA levels. The ABA mutants also showed reduced expression of genes involved in cutin or wax formation. This difference was again countered by exogenous ABA, further indicating regulation of cuticle biosynthesis by ABA. The fruit cuticles were affected differently by the ABA-associated mutations, but in general were thicker. However, no structural abnormalities were observed, and the cutin and wax compositions were less affected than in leaf cuticles, suggesting that ABA action influences cuticle formation in an organ-dependent manner. These results suggest dual roles for ABA in regulating leaf cuticle formation: one that is fundamentally associated with leaf expansion, independent of abiotic stress, and another that is drought induced.

Pea Border Cell Maturation and Release Involve Complex Cell Wall Structural Dynamics.

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.