Localisation and substrate specificities of transglycanases in charophyte algae relate to development and morphology.

Cell wall-modifying enzymes have been previously investigated in charophyte green algae (CGA) in cultures of uniform age, giving limited insight into their roles. Therefore, we investigated the in situ localisation and specificity of enzymes acting on hemicelluloses in CGA genera of different morphologies and developmental stages. In vivo transglycosylation between xyloglucan and an endogenous donor in filamentous Klebsormidium and Zygnema was observed in longitudinal cell walls of young (1 month) but not old cells (1 year), suggesting that it has a role in cell growth. By contrast, in parenchymatous Chara, transglycanase action occurred in all cell planes. In Klebsormidium and Zygnema, the location of enzyme action mainly occurred in regions where xyloglucans and mannans, and to a lesser extent mixed-linkage ß-glucan (MLG), were present, indicating predominantly xyloglucan:xyloglucan endotransglucosylase (XET) activity. Novel transglycosylation activities between xyloglucan and xylan, and xyloglucan and galactomannan were identified in vitro in both genera. Our results show that several cell wall-modifying enzymes are present in CGA, and that differences in morphology and cell age are related to enzyme localisation and specificity. This indicates an evolutionary significance of cell wall modifications, as similar changes are known in their immediate descendants, the land plants. This article has an associated First Person interview with the first author of the paper.

Cell wall extensins in root-microbe interactions and root secretions.

Extensins are cell wall glycoproteins, belonging to the hydroxyproline-rich glycoprotein (HRGP) family, which are involved in many biological functions, including plant growth and defence. Several reviews have described the involvement of HRGPs in plant immunity but little focus has been given specifically to cell wall extensins. Yet, a large set of recently published data indicates that extensins play an important role in plant protection, especially in root-microbe interactions. Here, we summarise the current knowledge on this topic and discuss the importance of extensins in root defence. We first provide an overview of the distribution of extensin epitopes recognised by different monoclonal antibodies among plants and discuss the relevance of some of these epitopes as markers of the root defence response. We also highlight the implication of extensins in different types of plant interactions elicited by either pathogenic or beneficial micro-organisms. We then present and discuss the specific importance of extensins in root secretions, as these glycoproteins are not only found in the cell walls but are also released into the root mucilage. Finally, we propose a model to illustrate the impact of cell wall extensin on root secretions.

Comparative in situ analysis reveals the dynamic nature of sclerenchyma cell walls of the fern Asplenium rutifolium.

Background and Aims: A key structural adaptation of vascular plants was the evolution of specialized vascular and mechanical tissues, innovations likely to have generated novel cell wall architectures. While collenchyma is a strengthening tissue typically found in growing organs of angiosperms, a similar tissue occurs in the petiole of the fern Asplenium rutifolium. Methods: The in situ cell wall (ultra)structure and composition of this tissue was investigated and characterized mechanically as well as structurally through nano-indentation and wide-angle X-ray diffraction, respectively. Key Results: Structurally the mechanical tissue resembles sclerenchyma, while its biomechanical properties and molecular composition both share more characteristics with angiosperm collenchyma. Cell wall thickening only occurs late during cell expansion or after cell expansion has ceased. Conclusions: If the term collenchyma is reserved for walls that thicken during expansive growth, the mechanical tissue in A. rutifolium represents sclerenchyma that mimics the properties of collenchyma and has the ability to modify its mechanical properties through sclerification. These results support the view that collenchyma does not occur in ferns and most probably evolved in angiosperms.

Plant and algal structure: from cell walls to biomechanical function.

Plant and algal cell walls are complex biomaterials composed of stiff cellulose microfibrils embedded in a soft matrix of polysaccharides, proteins and phenolic compounds. Cell wall composition differs between taxonomic groups and different tissue types (or even at the sub-cellular level) within a plant enabling specific biomechanical properties important for cell/tissue function. Moreover, cell wall composition changes may be induced in response to environmental conditions. Plant structure, habit, morphology and internal anatomy are also dependent on the taxonomic group as well as abiotic and biotic factors. This review aims to examine the complex and incompletely understood interactions of cell wall composition, plant form and biomechanical function.

Stomatal cell wall composition: distinctive structural patterns associated with different phylogenetic groups.

Background and Aims: Stomatal morphology and function have remained largely conserved throughout ∼400 million years of plant evolution. However, plant cell wall composition has evolved and changed. Here stomatal cell wall composition was investigated in different vascular plant groups in attempt to understand their possible effect on stomatal function. Methods: A renewed look at stomatal cell walls was attempted utilizing digitalized polar microscopy, confocal microscopy, histology and a numerical finite-elements simulation. The six species of vascular plants chosen for this study cover a broad structural, ecophysiological and evolutionary spectrum: ferns ( Asplenium nidus and Platycerium bifurcatum ) and angiosperms ( Arabidopsis thaliana and Commelina erecta ) with kidney-shaped stomata, and grasses (angiosperms, family Poaceae) with dumbbell-shaped stomata ( Sorghum bicolor and Triticum aestivum ). Key Results: Three distinct patterns of cellulose crystallinity in stomatal cell walls were observed: Type I (kidney-shaped stomata, ferns), Type II (kidney-shaped stomata, angiosperms) and Type III (dumbbell-shaped stomata, grasses). The different stomatal cell wall attributes investigated (cellulose crystallinity, pectins, lignin, phenolics) exhibited taxon-specific patterns, with reciprocal substitution of structural elements in the end-walls of kidney-shaped stomata. According to a numerical bio-mechanical model, the end walls of kidney-shaped stomata develop the highest stresses during opening. Conclusions: The data presented demonstrate for the first time the existence of distinct spatial patterns of varying cellulose crystallinity in guard cell walls. It is also highly intriguing that in angiosperms crystalline cellulose appears to have replaced lignin that occurs in the stomatal end-walls of ferns serving a similar wall strengthening function. Such taxon-specific spatial patterns of cell wall components could imply different biomechanical functions, which in turn could be a consequence of differences in environmental selection along the course of plant evolution.

Immunolocalization of cell wall carbohydrate epitopes in seaweeds: presence of land plant epitopes in Fucus vesiculosus L. (Phaeophyceae).

MAIN CONCLUSION: Land plant cell wall glycan epitopes are present in Fucus vesiculosus. RG-I/AG mAbs recognize distinct glycan epitopes in structurally different galactans, and 3-linked glucans are also present in the cell walls. Cell wall-directed monoclonal antibodies (mAbs) have given increased knowledge of fundamental land plant processes but are not extensively used to study seaweeds. We profiled the brown seaweed Fucus vesiculosus glycome employing 155 mAbs that recognize predominantly vascular plant cell wall glycan components. The resulting profile was used to inform in situ labeling studies. Several of the mAbs recognized and bound to epitopes present in different thallus parts of Fucus vesiculosus. Antibodies recognizing arabinogalactan epitopes were divided into four groups based on their immunolocalization patterns. Group 1 bound to the stipe, blade, and receptacles. Group 2 bound to the antheridia, oogonia and paraphyses. Group 3 recognized antheridia cell walls and Group 4 localized on the antheridia inner wall and oogonia mesochite. This study reveals that epitopes present in vascular plant cell walls are also present in brown seaweeds. Furthermore, the diverse in situ localization patterns of the RG-I/AG clade mAbs suggest that these mAbs likely detect distinct epitopes present in structurally different galactans. In addition, 3-linked glucans were also detected throughout the cell walls of the algal tissues, using the ß-glucan-directed LAMP mAb. Our results give insights into cell wall evolution, and diversify the available tools for the study of brown seaweed cell walls.

Pectin metabolism and assembly in the cell wall of the charophyte green alga Penium margaritaceum.

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.

Disruption of the microtubule network alters cellulose deposition and causes major changes in pectin distribution in the cell wall of the green alga, Penium margaritaceum.

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.

Plant and algal cell walls: diversity and functionality.

BACKGROUND: 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. SCOPE: 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.

Arabinogalactan protein-rich cell walls, paramural deposits and ergastic globules define the hyaline bodies of rhinanthoid Orobanchaceae haustoria.

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.

Comparative glycan profiling of Ceratopteris richardii 'C-Fern' gametophytes and sporophytes links cell-wall composition to functional specialization.

BACKGROUND AND AIMS: Innovations in vegetative and reproductive characters were key factors in the evolutionary history of land plants and most of these transformations, including dramatic changes in life cycle structure and strategy, necessarily involved cell-wall modifications. To provide more insight into the role of cell walls in effecting changes in plant structure and function, and in particular their role in the generation of vascularization, an antibody-based approach was implemented to compare the presence and distribution of cell-wall glycan epitopes between (free-living) gametophytes and sporophytes of Ceratopteris richardii 'C-Fern', a widely used model system for ferns. METHODS: Microarrays of sequential diamino-cyclohexane-tetraacetic acid (CDTA) and NaOH extractions of gametophytes, spores and different organs of 'C-Fern' sporophytes were probed with glycan-directed monoclonal antibodies. The same probes were employed to investigate the tissue- and cell-specific distribution of glycan epitopes. KEY RESULTS: While monoclonal antibodies against pectic homogalacturonan, mannan and xyloglucan widely labelled gametophytic and sporophytic tissues, xylans were only detected in secondary cell walls of the sporophyte. The LM5 pectic galactan epitope was restricted to sporophytic phloem tissue. Rhizoids and root hairs showed similarities in arabinogalactan protein (AGP) and xyloglucan epitope distribution patterns. CONCLUSIONS: The differences and similarities in glycan cell-wall composition between 'C-Fern' gametophytes and sporophytes indicate that the molecular design of cell walls reflects functional specialization rather than genetic origin. Glycan epitopes that were not detected in gametophytes were associated with cell walls of specialized tissues in the sporophyte.

Heterogeneity of silica and glycan-epitope distribution in epidermal idioblast cell walls in Adiantum raddianum laminae.

Laminae of Adiantum raddianum Presl., a fern belonging to the family Pteridaceae, are characterised by the presence of epidermal fibre-like cells under the vascular bundles. These cells were thought to contain silica bodies, but their thickened walls leave no space for intracellular silica suggesting it may actually be deposited within their walls. Using advanced electron microscopy in conjunction with energy dispersive X-ray microanalysis we showed the presence of silica in the cell walls of the fibre-like idioblasts. However, it was specifically localised to the outer layers of the periclinal wall facing the leaf surface, with the thick secondary wall being devoid of silica. Immunocytochemical experiments were performed to ascertain the respective localisation of silica deposition and glycan polymers. Epitopes characteristic for pectic homogalacturonan and the hemicelluloses xyloglucan and mannan were detected in most epidermal walls, including the silica-rich cell wall layers. The monoclonal antibody, LM6, raised against pectic arabinan, labelled the silica-rich primary wall of the epidermal fibre-like cells and the guard cell walls, which were also shown to contain silica. We hypothesise that the silicified outer wall layers of the epidermal fibre-like cells support the lamina during cell expansion prior to secondary wall formation. This implies that silicification does not impede cell elongation. Although our results suggest that pectic arabinan may be implicated in silica deposition, further detailed analyses are needed to confirm this. The combinatorial approach presented here, which allows correlative screening and in situ localisation of silicon and cell wall polysaccharide distribution, shows great potential for future studies.

Evolution and diversity of green plant cell walls.

Plant cells are surrounded by a dynamic cell wall that performs many essential biological roles, including regulation of cell expansion, the control of tissue cohesion, ion-exchange and defence against microbes. Recent evidence shows that the suite of polysaccharides and wall proteins from which the plant cell wall is composed shows variation between monophyletic plant taxa. This is likely to have been generated during the evolution of plant groups in response to environmental stress. Understanding the natural variation and diversity that exists between cell walls from different taxa is key to facilitating their future exploitation and manipulation, for example by increasing lignocellulosic content or reducing its recalcitrance for use in biofuel generation.

Localization, Extraction, and Quantification of Plant and Algal Arabinogalactan Proteins.

Arabinogalactan proteins are a diverse group of cell wall-associated proteoglycans. While structural and molecular genetic analyses have contributed to the emerging improved understanding of the wide-range of biological processes in which AGPs are implicated; the ability to detect, localize, and quantify them is fundamentally important. This chapter describes three methods: histological staining, radial gel diffusion, and colorimetric quantification, each of which utilize the ability of Yariv reagent to bind to AGPs.

Solanales Stem Biomechanical Properties Are Primarily Determined by Morphology Rather Than Internal Structural Anatomy and Cell Wall Composition.

Self-supporting plants and climbers exhibit differences in their structural and biomechanical properties. We hypothesized that such fundamental differences originate at the level of the material properties. In this study, we compared three non-woody members of the Solanales exhibiting different growth habits: (1) a self-supporting plant (potato, Solanum tuberosum), (2) a trailing plant (sweet potato, Ipomoea batatas), and (3) a twining climber (morning glory, Ipomoea tricolor). The mechanical properties investigated by materials analyses were combined with structural, biochemical, and immunohistochemical analyses. Generally, the plants exhibited large morphological differences, but possessed relatively similar anatomy and cell wall composition. The cell walls were primarily composed of hemicelluloses (~60%), with α-cellulose and pectins constituting ~25% and 5%-8%, respectively. Immunohistochemistry of specific cell wall components suggested only minor variation in the occurrence and localization between the species, although some differences in hemicellulose distribution were observed. According to tensile and flexural tests, potato stems were the stiffest by a significant amount and the morning glory stems were the most compliant and showed differences in two- and three-orders of magnitude; the differences between their effective Young's (Elastic) modulus values (geometry-independent parameter), on the other hand, were substantially lower (at the same order of magnitude) and sometimes not even significantly different. Therefore, although variability exists in the internal anatomy and cell wall composition between the different species, the largest differences were seen in the morphology, which appears to be the primary determinant of biomechanical function. Although this does not exclude the possibility of different mechanisms in other plant groups, there is apparently less constraint to modifying stem morphology than anatomy and cell wall composition within the Solanales.

Insights into the Evolution of Multicellularity from the Sea Lettuce Genome.

We report here the 98.5 Mbp haploid genome (12,924 protein coding genes) of Ulva mutabilis, a ubiquitous and iconic representative of the Ulvophyceae or green seaweeds. Ulva's rapid and abundant growth makes it a key contributor to coastal biogeochemical cycles; its role in marine sulfur cycles is particularly important because it produces high levels of dimethylsulfoniopropionate (DMSP), the main precursor of volatile dimethyl sulfide (DMS). Rapid growth makes Ulva attractive biomass feedstock but also increasingly a driver of nuisance "green tides." Ulvophytes are key to understanding the evolution of multicellularity in the green lineage, and Ulva morphogenesis is dependent on bacterial signals, making it an important species with which to study cross-kingdom communication. Our sequenced genome informs these aspects of ulvophyte cell biology, physiology, and ecology. Gene family expansions associated with multicellularity are distinct from those of freshwater algae. Candidate genes, including some that arose following horizontal gene transfer from chromalveolates, are present for the transport and metabolism of DMSP. The Ulva genome offers, therefore, new opportunities to understand coastal and marine ecosystems and the fundamental evolution of the green lineage.

Permanently open stomata of aquatic angiosperms display modified cellulose crystallinity patterns.

Most floating aquatic plants have stomata on their upper leaf surfaces, and usually their stomata are permanently open. We previously identified 3 distinct crystallinity patterns in stomatal cell walls, with angiosperm kidney-shaped stomata having the highest crystallinity in the polar end walls as well as the adjacent polar regions of the guard cells. A numerical bio-mechanical model suggested that the high crystallinity areas are localized to regions where the highest stress is imposed. Here, stomatal cell wall crystallinity was examined in 4 floating plants from 2 different taxa: basal angiosperms from the ANITA grade and monocots. It appears that the non-functional stomata of floating plants display reduced crystallinity in the polar regions as compared with high crystallinity of the ventral (inner) walls. Thus their guard cells are both less flexible and less stress resistant. Our findings suggest that the pattern of cellulose crystallinity in stomata of floating plants from different families was altered as a consequence of similar evolutionary pressures.

ß-1,3-Glucans are components of brown seaweed (Phaeophyceae) cell walls.

LAMP is a cell wall-directed monoclonal antibody (mAb) that recognizes a ß-(1,3)-glucan epitope. It has primarily been used in the immunolocalization of callose in vascular plant cell wall research. It was generated against a brown seaweed storage polysaccharide, laminarin, although it has not often been applied in algal research. We conducted in vitro (glycome profiling of cell wall extracts) and in situ (immunolabeling of sections) studies on the brown seaweeds Fucus vesiculosus (Fucales) and Laminaria digitata (Laminariales). Although glycome profiling did not give a positive signal with the LAMP mAb, this antibody clearly detected the presence of the ß-(1,3)-glucan in situ, showing that this epitope is a constituent of these brown algal cell walls. In F. vesiculosus, the ß-(1,3)-glucan epitope was present throughout the cell walls in all thallus parts; in L. digitata, the epitope was restricted to the sieve plates of the conductive elements. The sieve plate walls also stained with aniline blue, a fluorochrome used as a probe for callose. Enzymatic digestion with an endo-ß-(1,3)-glucanase removed the ability of the LAMP mAb to label the cell walls. Thus, ß-(1,3)-glucans are structural polysaccharides of F. vesiculosus cell walls and are integral components of the sieve plates in these brown seaweeds, reminiscent of plant callose.

Two sides of the same coin: Xyloglucan endotransglucosylases/hydrolases in host infection by the parasitic plant Cuscuta.

The holoparasitic angiosperm Cuscuta develops haustoria that enable it to feed on other plants. Recent findings corroborate the long-standing theory that cell wall modifications are required in order for the parasite to successfully infect a host, and further suggest that changes to xyloglucan through the activity of xyloglucan endotransglucosylases/hydrolases (XTHs) are essential. On the other hand, XTH expression was also detected in resistant tomato upon an attack by Cuscuta, which suggests that both host and parasite use these enzymes in their "arms race." Here, we summarize existing data on the cell wall-modifying activities of XTHs during parasitization and present a model suggesting how XTHs might function to make the host's resources accessible to Cuscuta.