Primary Plant Cell Wall Research Articles

Do Cell Wall Esters Facilitate Forest Response to Climate?

Terrestrial ecosystem dynamics are strongly modified by stresses associated with climate change, impacting plant growth and development, mortality, and ecological succession. Here we highlight the potential role of plant cell wall esters to link changes in cell wall structure and function with biosphere-atmosphere fluxes of methanol, acetic acid, carbon dioxide (CO2), and water (H2O).

Xyloglucan Is Not Essential for the Formation and Integrity of the Cellulose Network in the Primary Cell Wall Regenerated from Arabidopsis Protoplasts.

The notion that xyloglucans (XG) play a pivotal role in tethering cellulose microfibrils in the primary cell wall of plants can be traced back to the first molecular model of the cell wall proposed in 1973, which was reinforced in the 1990s by the identification of Xyloglucan Endotransglucosylase/Hydrolase (XTH) enzymes that cleave and reconnect xyloglucan crosslinks in the cell wall. However, this tethered network model has been seriously challenged since 2008 by the identification of the Arabidopsis thaliana xyloglucan-deficient mutant (xxt1 xxt2), which exhibits functional cell walls. Thus, the molecular mechanism underlying the physical integration of cellulose microfibrils into the cell wall remains controversial. To resolve this dilemma, we investigated the cell wall regeneration process using mesophyll protoplasts derived from xxt1 xxt2 mutant leaves. Imaging analysis revealed only a slight difference in the structure of cellulose microfibril network between xxt1 xxt2 and wild-type (WT) protoplasts. Additionally, exogenous xyloglucan application did not alter the cellulose deposition patterns or mechanical stability of xxt1 xxt2 mutant protoplasts. These results indicate that xyloglucan is not essential for the initial assembly of the cellulose network, and the cellulose network formed in the absence of xyloglucan provides sufficient tensile strength to the primary cell wall regenerated from protoplasts.

Fluorescence visualization of cellulose and pectin in the primary plant cell wall.

Plant cell walls constitute the extracellular matrix surrounding plant cells and are composed mainly of polysaccharides. The chemical makeup of the primary plant cell wall, and specifically, the abundance, localization and arrangement of the constituting polysaccharides are intimately linked with growth, morphogenesis and differentiation in plant cells. Visualization of the cell wall components is, therefore, a crucial tool in plant cell developmental studies. In this technical update, we present protocols for fluorescence visualization of cellulose and pectin in selected plant tissues and illustrate examples of some of the available labels that hold promise for live imaging of plant cell wall expansion and morphogenesis.

Identification of two functional xyloglucan galactosyltransferase homologs BrMUR3 and BoMUR3 in brassicaceous vegetables.

Xyloglucan (XyG) is the predominant hemicellulose in the primary cell walls of most dicotyledonous plants. Current models of these walls predict that XyG interacts with cellulose microfibrils to provide the wall with the rigidity and strength necessary to maintain cell integrity. Remodeling of this network is required to allow cell elongation and plant growth. In this study, homologs of Arabidopsis thaliana MURUS3 (MUR3), which encodes a XyG-specific galactosyltransferase, were obtained from Brassica rapa (BrMUR3) to Brassica oleracea (BoMUR3). Genetic complementation showed that BrMUR3 and BoMUR3 rescue the phenotypic defects of the mur3-3 mutant. Xyloglucan subunit composition analysis provided evidence that BrMUR3 and BoMUR3 encode a galactosyltransferase, which transfers a galactose residue onto XyG chains. The detection of XXFG and XLFG XyG subunits (restoration of fucosylated side chains) in mur3-3 mutants overexpressing BrMUR3 or BoMUR3 show that MUR3 from Brassica to Arabidopsis are comparable as they add Gal to the third xylosyl residue of the XXXG subunit. Our results provide additional information for functional dissection and evolutionary analysis of MUR3 genes derived from brassicaceous species.

The Living Fossil Psilotum nudum Has Cortical Fibers With Mannan-Based Cell Wall Matrix.

Cell wall thickening and development of secondary cell walls was a major step in plant terrestrialization that provided the mechanical support, effective functioning of water-conducting elements and fortification of the surface tissues. Despite its importance, the diversity, emergence and evolution of secondary cell walls in early land plants have been characterized quite poorly. Secondary cell walls can be present in different cell types with fibers being among the major ones. The necessity for mechanical support upon increasing plant height is widely recognized; however, identification of fibers in land plants of early taxa is quite limited. In an effort to partially fill this gap, we studied the fibers and the composition of cell walls in stems of the sporophyte of the living fossil Psilotum nudum. Various types of light microscopy, combined with partial tissue maceration demonstrated that this perennial, rootless, fern-like vascular plant, has abundant fibers located in the middle cortex. Extensive immunodetection of cell wall polymers together with various staining and monosaccharide analysis of cell wall constituents revealed that in P. nudum, the secondary cell wall of its cortical fibers is distinct from that of its tracheids. Primary cell walls of all tissues in P. nudum shoots are based on mannan, which is also common in other extant early land plants. Besides, the primary cell wall contains epitope for LM15 specific for xyloglucan and JIM7 that binds methylesterified homogalacturonans, two polymers common in the primary cell walls of higher plants. Xylan and lignin were detected as the major polymers in the secondary cell walls of P. nudum tracheids. However, the secondary cell wall in its cortical fibers is quite similar to their primary cell walls, i.e., enriched in mannan. The innermost secondary cell wall layer of its fibers but not its tracheids has epitope to bind the LM15, LM6, and LM5 antibodies recognizing, respectively, xyloglucan, arabinan and galactan. Together, our data provide the first description of a mannan-based cell wall in sclerenchyma fibers, and demonstrate in detail that the composition and structure of secondary cell wall in early land plants are not uniform in different tissues.

Anisotropic Motions of Fibrils Dictated by Their Orientations in the Lamella: A Coarse-Grained Model of a Plant Cell Wall.

Plant cell walls are complex systems that exhibit the characteristics of both rigid and soft material depending on their external perturbations. The three main polymeric components in a plant primary cell wall are cellulose fibrils, hemicellulose, and pectins. These components interact in a hierarchical fashion giving rise to mesoscale structural features such as cellulose bundles, lamella stacking, and so on. Although several studies have focused on understanding these unique structural features, a clear picture linking them to cell wall mechanics is still lacking. As a first step toward this goal, a phenomenological model of plant cell wall has been developed in this work by using available experimental data to investigate the underlying connections between mesoscale structural features and the motions of fibrils during deformation. In this model cellulose fibrils exhibit motions such as angular reorientations and kinking upon forced stretching. These motions are dependent on the orientation of fibrils with respect to the stretch direction, i.e., fibrils that are at an angle to the stretch direction exhibit predominant angular reorientations, while fibrils transverse to the stretch direction undergo kinking as a result of transverse compression. Varying the chain length of pectin had negligible effects on these motions. One of the main contributions from this work is the development of a simple model that can be easily fine-tuned to test other hypotheses and extended to include additional experimental knowledge about the structural aspects of cell walls in the future.

The contribution of cell wall remodeling and signaling to lateral organs formation

Lateral organs are formed in plants by post embryonic developmental programs. Leaves, and flowers differentiate from the shoot apical meristem and lateral roots from the primary root pericycle meristem. Adventitious roots are roots formed from non-root lateral meristematic tissues, mostly the cambium, in many cases in response to stress signals. The ability of plants to regenerate adventitious roots is fundamental for selection and breading programs which are based on vegetative propagation of elite clones. Thus, recalcitrant plants, losing their rooting capability, may form a genuine commercial barrier in agricultural and forestry improvement programs. Some cellular mechanisms underlying adventitious root formation have been revealed, but much is yet to be clarified. The plant primary cell wall is a dynamic organ that can change its form, and perceive and relay molecular signals inward and outward during certain stages of development in particular cells. Therefore, before the secondary cell wall is deposited and plants become the wood from which walls and furniture are built, and the fibers from which cloths are woven, primary cell walls actively participate in plant cell differentiation and developmental programs. While auxin is a major regulator, cell walls are important in regulating coherent formative cell division and synchronized polar elongation of cell lineages that are necessary for lateral organ induction and formation, and collaborative cell functioning. Nevertheless, little is known of how cell wall changes are molecularly sensed and translated to intracellular signals during differentiation of adventitious roots. Here we summarize recent data linking, directly or indirectly, cell wall events to auxin signaling and to lateral or adventitious root induction and formation.

Interaction between Autonomous and Microtubule Guidance Systems Controls Cellulose Synthase Trajectories.

The organization of cellulose microfibrils is critical for the strength and growth of plant cell walls. Microtubules have been shown to play a key role in controlling microfibril organization by guiding cellulose synthase complexes [1-4]. However, cellulose synthase trajectories can be maintained when microtubules are removed by drugs, suggesting a separate guidance mechanism is also at play [1, 5, 6]. By slowing down microtubule dynamics, we reveal such a mechanism by showing that cellulose synthase complexes can interact with the trails left by other complexes, causing them to follow the trails or disappear. The stability of the trails, together with the sensitivity of their directions to cellulase treatment, indicates they most likely reflect nascent cellulose microfibrils. Over many hours, this autonomous mechanism alone can lead to a change in the dominant orientation of cellulose synthase trajectories. However, the mechanism can be overridden by the microtubule guidance system. Our findings suggest a dual guidance model, in which an autonomous system, involving interaction between cellulose synthases and microfibrils, can maintain aligned cellulose synthase trajectories, while a microtubule guidance system allows alignments to be steered by environmental and developmental cues.

Using Solid-State 13C NMR Spectroscopy to Study the Molecular Organization of Primary Plant Cell Walls.

A knowledge of the mobilities of the polysaccharides or parts of polysaccharides in a cell-wall preparation provides information about possible molecular interactions among the polysaccharides in the cell wall and the relative locations of polysaccharides within the cell wall. A number of solid-state 13C NMR techniques have been developed that can be used to investigate different types of polysaccharide mobilities: rigid, semirigid, mobile, and highly mobile. In this chapter techniques are described for obtaining spectra from primary cell-wall preparations using CP/MAS, proton-rotating frame, proton spin-spin, spin-echo relaxation spectra and single-pulse excitation. We also describe how proton spin relaxation editing can be used to obtain subspectra for cell-wall polysaccharides of different mobilities, and how 2D and 3D solid-state NMR experiments have recently been applied to plant cell walls.

MUR1-mediated cell-wall fucosylation is required for freezing tolerance in Arabidopsis thaliana.

Summary Forward genetic screens play a key role in the identification of genes contributing to plant stress tolerance. Using a screen for freezing sensitivity, we have identified a novel freezing tolerance gene, SENSITIVE‐TO‐FREEZING8, in Arabidopsis thaliana. We identified SFR8 using recombination‐based mapping and whole‐genome sequencing. As SFR8 was predicted to have an effect on cell wall composition, we used GC‐MS and polyacrylamide gel electrophoresis to measure cell‐wall fucose and boron (B)‐dependent dimerization of the cell‐wall pectic domain rhamnogalacturonan II (RGII) in planta. After treatments to promote borate‐bridging of RGII, we assessed freeze‐induced damage in wild‐type and sfr8 plants by measuring electrolyte leakage from freeze‐thawed leaf discs.We mapped the sfr8 mutation to MUR1, a gene encoding the fucose biosynthetic enzyme GDP‐d‐mannose‐4,6‐dehydratase. sfr8 cell walls exhibited low cell‐wall fucose levels and reduced RGII bridging. Freezing sensitivity of sfr8 mutants was ameliorated by B supplementation, which can restore RGII dimerization. B transport mutants with reduced RGII dimerization were also freezing‐sensitive.Our research identifies a role for the structure and composition of the plant primary cell wall in determining basal plant freezing tolerance and highlights the specific importance of fucosylation, most likely through its effect on the ability of RGII pectin to dimerize.

Mobility of pectin methylesterase in pectin/cellulose gels is enhanced by the presence of cellulose and by its catalytic capacity

The pectin methylesterase action is usually studied in a homogeneous aqueous medium in the presence of a large excess of soluble substrate and water. However in the cell wall, the water content is much lower, the substrate is cross-linked with itself or with other polymers, and the enzyme has to diffuse through the solid matrix before catalysing the linkage breakdown. As plant primary cell walls can be considered as cellulose-reinforced hydrogels, this study investigated the diffusion of a fungal pectin methylesterase in pectin/cellulose gels used as cell wall-mimicking matrix to understand the impact of this matrix and its (micro) structure on the enzyme’s diffusion within it. The enzyme mobility was followed by synchrotron microscopy thanks to its auto-fluorescence after deep-UV excitation. Time-lapse imaging and quantification of intensity signal by image analysis revealed that the diffusion of the enzyme was impacted by at least two criteria: (i) only the active enzyme was able to diffuse, showing that the mobility was related to the catalytic ability, and (ii) the diffusion was improved by the presence of cellulose in the gel.

A REVIEW OF WOOD-BARK ADHESION: METHODS AND MECHANICS OF DEBARKING FOR WOODY BIOMASS

Debarking systems and strategies are used to increase the value of woody biomass by separating wood and bark into two value-added product streams. Several debarking methods have been used for the removal of bark from wood. The selection of a debarking method is often based on the wood type and its end use. Debarking methods discussed briefly in this review include drum debarking, ring debarking, cradle debarking, chain flail debarking, high-pressure water jet debarking, compression debarking, and biodebarking. The performance of these debarking methods is highly dependent on operational parameters of machines, properties and type of woody biomass, and pretreatments. A universal applicable mechanistic model of the debarking process would be especially valuable for the development and optimization of debarking systems. In this respect, the competing objectives of high bark removal, low wood damage, high throughput, and low energy must be balanced against one another to arrive at a truly optimized approach. Wood-bark bond strength plays a vital role in impacting the effectiveness and efficiency of debarking technology. Thus, it is important to understand how mechanical properties of the wood-bark interface are influenced by different factors. Key factors that affect the wood-bark bond strength include MC, harvest season, wood species, temperature, and direction of applied load. The likely reason why MC affects the wood-bark adhesion strength is that the constituent elements of primary plant cell walls (cellulose, hemicellulose, and pectin) behave differently when they are exposed to water molecules. For example, there is negligible change in the length if cellulose microfibrils when exposed to water molecules. However, characteristics of hemicellulose and pectin behave differently than cellulose when they contact with water molecules. Also, the difference in the adhesion strength of wood-bark bond among varieties of woody plants is possibly due to the difference in density of cross linkages of homogalacturonan pectin by Ca2., and arabinan and galactan side chains of pectin. The relevance of this information to the debarking process is discussed.

Modeling the nonlinear elastic behavior of plant epidermis

Cell growth and organ development in plants are often correlated with the tensile behavior of the primary cell wall. To understand the mechanical behavior of plant material, various mechanical testing techniques have been employed, such as tensile testing of excised tissue samples. The onion (Allium cepa L.) epidermis has emerged as a model system for plant tissue mechanics. In this study, we performed tensile tests on strips of adaxial onion epidermis. While the tissue appeared stiffer in the direction along the major growth axis compared with the transverse direction, the tensile strength of tissue was not significantly different between the two orientations, indicating a nontrivial link between the cell wall and tissue mechanical anisotropy. Importantly, we observed the stress–strain behavior of the onion epidermis under tension to be highly nonlinear. Several hyperelastic models were fitted to the test data to evaluate their capacity to describe the nonlinear deformation of onion epidermis. The Yeoh hyperelastic model could successfully simulate the uniaxial tensile test data. This study suggests that accounting for nonlinearity in the deformation of the primary tissue may be essential for the accurate interpretation of mechanical test data, and a better understanding of the mechanics of the primary plant cell wall.

Nanostructure and poroviscoelasticity in cell wall materials from onion, carrot and apple: Roles of pectin

The hierarchical organisation of polysaccharides in primary plant cell walls is responsible for their unique mechanical properties, and in turn for the textural and rheological properties of plant-based foods and ingredients. It is expected that at the nano scale, the mechanical properties of cell wall materials arise from a combination of structural deformation of the polysaccharide networks and hydraulic properties of the continuous water phase, as has been shown for other cellulose-based composites. Pectin plays a key role in the load bearing properties of (bacterial) cellulose-pectin composites due to its contribution to both hydration structure and the dynamics of water movement. To investigate whether these features are also important in plant cell wall materials we have used a set of advanced characterisation techniques to elucidate cell wall structural features at different length scales (X-ray diffraction and small angle X-ray and neutron scattering) in cell walls from two dicotyledons (apple and carrot) and a non-commelinid monocotyledon (onion). The strength of isolated cell walls was measured under compression and fitted to a poroviscoelastic mechanical model, demonstrating that the mechanical properties of the isolated cell wall materials are directly linked to both polysaccharide networks and fluid flow through the networks. Our results show how pectin polysaccharides influence the viscoelastic behaviour of these materials and contribute to the texture of plant-derived food systems.

Methods to quantify primary plant cell wall mechanics.

The primary plant cell wall is a dynamically regulated composite material of multiple biopolymers that forms a scaffold enclosing the plant cells. The mechanochemical make-up of this polymer network regulates growth, morphogenesis, and stability at the cell and tissue scales. To understand the dynamics of cell wall mechanics, and how it correlates with cellular activities, several experimental frameworks have been deployed in recent years to quantify the mechanical properties of plant cells and tissues. Here we critically review the application of biomechanical tool sets pertinent to plant cell mechanics and outline some of their findings, relevance, and limitations. We also discuss methods that are less explored but hold great potential for the field, including multiscale in silico mechanical modeling that will enable a unified understanding of the mechanical behavior across the scales. Our overview reveals significant differences between the results of different mechanical testing techniques on plant material. Specifically, indentation techniques seem to consistently report lower values compared with tensile tests. Such differences may in part be due to inherent differences among the technical approaches and consequently the wall properties that they measure, and partly due to differences between experimental conditions.

Genome-Wide Identification and Expression Profiling of the Polygalacturonase (PG) and Pectin Methylesterase (PME) Genes in Grapevine (Vitis vinifera L.).

In pectin regulation, polygalacturonases (PGs) and pectin methylesterases (PMEs) are critical components in the transformation, disassembly network, and remodeling of plant primary cell walls. In the current study, we identified 36 PG and 47 PME genes using the available genomic resources of grapevine. Herein, we provide a comprehensive overview of PGs and PMEs, including phylogenetic and collinearity relationships, motif and gene structure compositions, gene duplications, principal component analysis, and expression profiling during developmental stages. Phylogenetic analysis of PGs and PMEs revealed similar domain composition patterns with Arabidopsis. The collinearity analysis showed high conservation and gene duplications with purifying selection. The type of duplications also varied in terms of gene numbers in PGs (10 dispersed, 1 proximal, 12 tandem, and 13 segmental, respectively) and PMEs (23 dispersed, 1 proximal, 16 tandem, and 7 segmental, respectively). The tissue-specific response of PG and PME genes based on the reported transcriptomic data exhibited diverged expression patterns in various organs during different developmental stages. Among PGs, VvPG8, VvPG10, VvPG13, VvPG17, VvPG18, VvPG19, VvPG20, VvPG22, and VvPG23 showed tissue- or organ-specific expression in majority of the tissues during development. Similarly, in PMEs, VvPME3, VvPME4, VvPME5, VvPME6, VvPME19, VvPME21, VvPME23, VvPME29, VvPME31, and VvPME32 suggested high tissue-specific response. The gene ontology (GO), Kyoto Encyclopedia of Genes and Genomics (KEGG) enrichment, and cis-elements prediction analysis also suggested the putative functions of PGs and PMEs in plant development, such as pectin and carbohydrate metabolism, and stress activities. Moreover, qRT-PCR validation of 32 PG and PME genes revealed their role in various organs of grapevines (i.e., root, stem, tendril, inflorescence, flesh, skins, and leaves). Therefore, these findings will lead to novel insights and encourage cutting-edge research on functional characterization of PGs and PMEs in fruit crop species.

Biobased Cellulose Nanofibril-Oil Composite Films for Active Edible Barriers.

Low-concentration oil-in-water emulsions stabilized by cellulose nanofibrils (CNFs) extracted from primary plant cell wall materials are used to prepare thin biobased CNF–oil composite films by solvent casting. Flexible, transparent, and biodegradable composite films are obtained, with increased thermal stability (up to 300 °C) as the oil concentration increases. Examination of the microstructure demonstrates a clear dependency on the oil content, as a multilayered structure where the oil phase trapped between two layers of CNFs is appreciated at high oil concentrations. The embedded oil significantly influences the mechanical and wetting properties of the films, confirming their potential for use in packaging systems. Encapsulation of curcumin in the composite films leads to an increased antioxidant (up to 30% radical scavenging activity) and antimicrobial activity, inhibiting the growth of foodborne bacteria such as Escherichia coli. The resulting composite films show promising results in the field of active packaging for applications in the food, pharmaceutical, and cosmetic industries.

Stronger cellulose microfibril network structure through the expression of cellulose-binding modules in plant primary cell walls

Cellulose-binding modules (CBMs) are non-catalytic domains typically occurring in glycoside hydrolases. Their specific interaction with diverse polysaccharides assists hydrolysis by the catalytic subunits. In this work, we have exploited the interactions between a CBM from family 3 (CBM3) and cell wall polysaccharides to alter the structure and mechanical properties of cellulose microfibrils from BY-2 tobacco cell suspension cultures. A CBM3 from Clostridium thermocellum was overexpressed in the cells using Agrobacterium-mediated transformation. Water suspensions of cellulose microfibrils were prepared by the removal of the non-cellulosic components of the primary cell walls, followed by mild disintegration using sonication. The morphology of the microfibrils was characterized by transmission electron microscopy and atomic force microscopy. These cellulose microfibrils were further hydrolyzed with 64 wt% sulfuric acid to produce cellulose nanocrystals (CNCs). The average length of CNCs prepared from the CBM3-transformed cells was 201 nm, higher than that from the wild-type cells (122 nm). In addition, the mechanical properties and deformation mechanism of nanopapers prepared from suspensions of cellulose microfibrils were investigated. The nanopapers obtained from the CBM3-transformed cells exhibited enhanced tensile strength and work of fracture, 40% and 128% higher than those prepared from wild-type tobacco cells, respectively.Graphical abstract

Arabinose substitution effect on xylan rigidity and self-aggregation

Substituted xylans play an important role in the structure and mechanics of the primary cell wall of plants. Arabinoxylans (AX) consist of a xylose backbone substituted with arabinose, while glucuronoarabinoxylans (GAX) also contain glucuronic acid substitutions and ferulic acid esters on some of the arabinoses. We provide a molecular-level description on the dependence of xylan conformational, self-aggregation properties and binding to cellulose on the degree of arabinose substitution. Molecular dynamics simulations reveal fully solubilized xylans with a low degree of arabinose substitution (lsAX) to be stiffer than their highly substituted (hsAX) counterparts. Small-angle neutron scattering experiments indicate that both wild-type hsAX and debranched lsAX form macromolecular networks that are penetrated by water. In those networks, lsAX are more folded and entangled than hsAX chains. Increased conformational entropy upon network formation for hsAX contributes to AX loss of solubility upon debranching. Furthermore, simulations show the intermolecular contacts to cellulose are not affected by arabinose substitution (within the margin of error). Ferulic acid is the GAX moiety found here to bind to cellulose most strongly, suggesting it may play an anchoring role to strengthen GAX-cellulose interactions. The above results suggest highly substituted GAX acts as a spacer, keeping cellulose microfibrils apart, whereas low substitution GAX is more localized in plant cell walls and promotes cellulose bundling.

Probing adhesion between nanoscale cellulose fibres using AFM lateral force spectroscopy: The effect of hemicelluloses on hydrogen bonding

Inter-fibre adhesion is a key contributing factor to the mechanical response and functionality of cellulose-based biomaterials. ‘Dip-and-Drag’ lateral force atomic force microscopy technique is used here to evaluate the influence of arabinoxylan and xyloglucan on interactions between nanoscale cellulose fibres within a hydrated network of bacterial cellulose. A cohesive zone model of the detachment event between two nano-fibres is used to interpret the experimental data and evaluate inter-fibre adhesion energy. The presence of xyloglucan or arabinoxylan is found to increase the adhesive energy by a factor of 4.3 and 1.3, respectively, which is consistent with these two hemicellulose polysaccharides having different specificity of hydrogen bonding with cellulose. Importantly, xyloglucan’s ability to strengthen adhesion between cellulose nano-fibres supports emergent models of the primary plant cell walls (Park & Cosgrove, 2012b), which suggest that xyloglucan chains confined within cellulose-cellulose junctions play a key role in cell wall’s mechanical response.