Tissue stresses in growing plant organs

Rapidly growing plant organs (e.g. coleopties, hypocotyls, or internodes) are composed of tissues that differ with respect to the thickness, structure, and extensibility of their cell walls. The thick, relatively inextensible outer wall of the epidermal cells contains both transverse and longitudinally oriented cellulose-microfibrils. The orientation of microfibrils of the thin, extensible walls of the parenchyma cells seems to be predominantly transverse. In many growing organs (i.e. leafstalks), the outer epidermal wall is supported by a thickened inner epidermal wall and by thick-walled subepidermal collenchyma tissue. Owing to the turgor pressure of the cells the peripheral walls are under tension, while the extensible inner tissue is under compression. As a corollary, the longitudinal tensile stress of the rigid peripheral wall is high whereas that of the internal walls is lowered. The physical stress between the tissues has been described by Sachs in 1865 as 'tissue tension'. The term 'tissue stress'. however, seems to be more appropriate since it comprises both tension and compression. Hitherto no method has been developed to measure tissue stresses directly as force per unit cross-sectional area. One can demonstrate the existence of tissue stresses by separation of the tissues (splitting, peeling) and determining the resulting strain of the isolated organ fragments. Based on such experiments it has been shown that rapid growth is always accompanied by the existence of longitudinal tissue stresses.     

Immunogold localization of xyloglucan and rhamnogalacturonan I in the cell walls of suspension-cultured sycamore cells

Plant cell walls serve several functions: they impart rigidity to the plant, provide a physical and chemical barrier between the cell and its environment, and regulate the size and shape of each cell. Chemical studies have provided information on the biochemical composition of the plant cell walls as well as detailed knowledge of individual cell wall molecules. In contrast, very little is known about the distribution of specific cell wall components around individual cells and throughout tissues. To address this problem, we have produced polyclonal antibodies against two cell wall matrix components; rhamnogalacturonan I (RG-I), a pectic polysaccharide, and xyloglucan (XG), a hemicellulose. By using the antibiodies as specific markers we have been able to localize these polymers on thin sections of suspension-cultured sycamore cells (Acer pseudoplatanus). Our results reveal that each molecule has a unique distribution. XG is localized throughout the entire wall and middle lamella. RG-I is restricted to the middle lamella and is especially evident in the junctions between cells. These observations indicate that plant cell walls may have more distinct chemical (and functional?) domains than previously envisaged.     

WAKs; cell wall associated kinases.

Students of metazoan biology have traditionally viewed the extracellular matrix (ECM) as a substrate with which cells interact to participate in developmental pattern formation and define a specific location. In contrast, the plant cell wall has been viewed as a cage that limits and thus directs plant cell morphology, and perhaps for this reason many have shied away from calling the plant cell wall the ECM. The recent discovery of a variety of receptor molecules and their ligands on the surface of plant cells and the intimate role cell walls play in development should direct our thinking toward a more dynamic view of the plant cell wall. A recent example, is the discovery of wall associated kinases (WAKs), which may well signal between the ECM and the cell and are required for cell expansion.     

Cell wall traits that influence plant development,immunity, and bioconversion

The architecture of the plant cell wall is highly dynamic, being substantially re‐modeled during growth and development. Cell walls determine the size and shape of cells and contribute to the functional specialization of tissues and organs. Beyond the physiological dynamics, the wall structure undergoes changes upon biotic or abiotic stresses. In this review several cell wall traits, mainly related to pectin, one of the major matrix components, will be discussed in relation to plant development, immunity and industrial bioconversion of biomass, especially for energy production. Plant cell walls are a source of oligosaccharide fragments with a signaling function for both development and immunity. Sensing cell wall damage, sometimes through the perception of released damage‐associated molecular patterns (DAMPs), is crucial for some developmental and immunity responses. Methodological advances that are expected to deepen our knowledge of cell wall (CW) biology will also be presented.     

毛竹茎细胞壁半纤维素多糖的免疫细胞化学定位研究

本文以毛竹(Phyllostachys pubescens)为材料,采用免疫细胞化学标记方法对两种细胞壁半纤维素多糖成分,即木聚糖(Xylan)和(1-3)(1-4)-β-葡聚糖［(1-3)(1-4)-β-glucan］在毛竹茎中的分布进行了观察。结果表明,应用免疫细胞化学方法可以准确、有效地观察这两种半纤维素多糖成分在细胞壁中的分布;木聚糖分布在已木质化的组织细胞的细胞壁中,与细胞壁木质化有密切关系;(1-3)(1-4)-β-葡聚糖在幼竹茎基本组织中分布于短薄壁细胞细胞壁中及长薄壁细胞胞间层,而在老龄竹茎基本组织中,仅分布于短薄壁细胞细胞壁中,而长薄壁细胞细胞壁却无此成分,反映出长、短薄壁细胞细胞壁组成上的差异。     

Electron Tomography of Cryo-Immobilized Plant Tissue: A Novel Approach to Studying 3D Macromolecular Architecture of Mature Plant Cell Walls In Situ

Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D) organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. In this paper we demonstrate that by cryo-immobilizing fresh tissue, then either cryo-sectioning or freeze-substituting and resin embedding, followed by cryo- or room temperature (RT) electron tomography, respectively, we can visualize previously unseen details of plant cell wall architecture in 3D, at macromolecular resolution (∼2 nm), and in near-native state. Qualitative and quantitative analyses showed that wall organization of cryo-immobilized samples were preserved remarkably better than conventionally prepared samples that suffer substantial extraction. Lignin-less primary cell walls were well preserved in both self-pressurized rapidly frozen (SPRF), cryo-sectioned samples as well as high-pressure frozen, freeze-substituted and resin embedded (HPF-FS-resin) samples. Lignin-rich secondary cell walls appeared featureless in HPF-FS-resin sections presumably due to poor stain penetration, but their macromolecular features could be visualized in unprecedented details in our cryo-sections. While cryo-tomography of vitreous tissue sections is currently proving to be instrumental in developing 3D models of lignin-rich secondary cell walls, here we confirm that the technically easier method of RT-tomography of HPF-FS-resin sections could be used immediately for routine study of low-lignin cell walls. As a proof of principle, we characterized the primary cell walls of a mutant (cob-6) and wild type Arabidopsis hypocotyl parenchyma cells by RT-tomography of HPF-FS-resin sections, and detected a small but significant difference in spatial organization of cellulose microfibrils in the mutant walls.     

Tissue-related changes in methyl-esterification of pectic polysaccharides in cauliflower (Brassica oleracea L. var. botrytis) stems

Pectic substances are a major component of cell walls in vegetable plants and have an important influence on plant food texture. Cauliflower (Brassica oleracea L. var. botrytis) stem sections at different regions of the mature plant stem have been monitored for tissue-related changes in the native pectic polysaccharides. Chemical analysis detected appreciable differences in the degree of methyl-esterification (ME) of pectic polysaccharides. About 65% of galacturonic acid (GalpA) residues were methyl-esterified in floret tissues. Relative ME showed a basipetal decrease, from 94% in the upper stem to 51% in the lower-stem vascular tissues. The decrease was not related to a basipetal increase in glucuronic acid (GlcpA) residues. The monoclonal antibodies, JIM 5 and JIM 7, produced distinct labelling patterns for the relatively low-methyl-esterified and high-methyl-esterified pectin epitopes, respectively. Labelling was related to cell type and tissue location in the stem. Floret cell walls contained epitopes for both JIM 5 and JIM 7 throughout the wall. Stem vascular tissues labelled more strongly with JIM 5. Whereas pith parenchyma in the upper stem labelled more strongly with JIM 7, in the lower-stem pith parenchyma, JIM 5 labelling predominated. Localization of pectic polysaccharide epitopes in cell walls provides an insight into how structural modifications might relate to the textural and nutritional properties of cell walls. Received: 16 August 1997 / Accepted: 20 December 1997     

Measuring the Deformation of Cells within a Piece of Compressed Potato Tuber Tissue

For the successful mathematical mechanical modelling of livingplant tissues, relationships between cellular deformations andtissue deformation need to be investigated. In previous workthese relationships have often been assumed. In this paper thedeformation of living cells within potato tuber tissue is measuredusing light microscopy and image analysis and is analysed inrelation to applied tissue deformations. The cell wall deformationwas found to depend upon the orientation of the cell wall faceswith respect to the global axes of the tissue and the appliedtissue deformation. Some faces experienced compression, whichreduced their surface area; others were deformed in bi-axialtension, thus increasing their surface area. These deformationswere successfully related to the global tissue deformations,using a simple constant volume affine deformation model, upto compressive deformations of 20% of specimen height. Somedeviation from the model was observed due to the bending ofcell walls in compression. Copyright 2000 Annals of Botany Company Potato tuber tissue, Solanum tuberosum, mechanical properties, cell walls, strain, re-orientation     

Mathematical modelling of the cellular mechanics of plants

The complex mechanical behaviour of plant tissues reflects the complexity of their structure and material properties. Modelling has been widely used in studies of how cell walls, single cells and tissue respond to loading, both externally applied loading and loads on the cell wall resulting from changes in the pressure within fluid-filled cells. This paper reviews what approaches have been taken to modelling and simulation of cell wall, cell and tissue mechanics, and to what extent models have been successful in predicting mechanical behaviour. Advances in understanding of cell wall ultrastructure and the control of cell growth present opportunities for modelling to clarify how growth-related mechanical properties arise from wall polymeric structure and biochemistry.     

Responses of Chrysanthemum Cells to Mechanical Stimulation Require Intact Microtubules and Plasma Membrane–Cell Wall Adhesion

Plant cells are highly susceptible and receptive to physical factors, both in nature and under experimental conditions. Exposure to mechanical forces dramatically results in morphological and microstructural alterations in their growth. In the present study, cells from chrysanthemum (Dendranthema morifolium) were subjected to constant pressure from an agarose matrix, which surrounded and immobilized the cells to form a cell-gel block. Cells in the mechanically loaded blocks elongated and divided, with an axis preferentially perpendicular to the direction of principal stress vectors. After a sucrose-induced plasmolysis, application of peptides containing an RGD motif, which interferes with plasma membrane-cell wall adhesion, reduced the oriented growth under stress conditions. Moreover, colchicines, but not cytochalasin B, abolished the effects of mechanical stress on cell morphology. Cellulose staining revealed that mechanical force reinforces the architecture of cell walls and application of mechanical force, and RGD peptides caused aggregative staining on the surface of plasmolyzed protoplasts. These results provide evidence that the oriented cell growth in response to compressive stress requires the maintenance of plasmalemma-cell wall adhesion and intact microtubules. Stress-triggered wall development in individual plant cells was also demonstrated.     

Macromolecular biophysics of the plant cell wall: Concepts and methodology

Plant cell walls provide form and mechanical strength to the living plant, but the relationship between their complex architecture and their remarkable ability to withstand external stress is not well understood. Primary cell walls are adapted to withstand tensile stresses while secondary cell walls also need to withstand compressive stresses. Therefore, while primary cell walls can with advantage be flexible and elastic, secondary cell walls must be rigid to avoid buckling under compressive loads. In addition, primary cell walls must be capable of growth and are subjected to cell separation forces at the cell corners. To understand how these stresses are resisted by cell walls, it will be necessary to find out how the walls deform internally under load, and how rigid are specific constituents of each type of cell wall. The most promising spectroscopic techniques for this purpose are solid-state nuclear magnetic resonance (NMR), and Fourier-transform infrared (FTIR) and Raman microscopy. By NMR relaxation experiments, it is possible to probe thermal motion in each cell-wall component. Novel adaptations of FTIR and Raman spectroscopy promise to allow mechanical stress and strain upon specific polymers to be examined in situ within the cell wall.     

What makes plants different? Principles of extracellular matrix function in 'soft' plant tissues

An overview of the biomechanic and morphogenetic function of the plant extracellular matrix (ECM) in its primary state is given. ECMs can play a pivotal role in cellular osmo- and volume-regulation, if they enclose the cell hermetically and constrain hydrostatic pressure evoked by osmotic gradients between the cell and its environment. From an engineering viewpoint, such cell walls turn cells into hydraulic machines, which establishes a crucial functional differences between cell walls and other cellular surface structures. Examples of such hydraulic machineries are discussed. The function of cell walls in the control of pressure, volume, and shape establishes constructional evolutionary constraints, which can explain aspects commonly considered typical of plants (sessility, autotrophy). In plants, 'cell division' by insertion of a new cell wall is a process of internal cytoplasmic differentiation. As such it differs fundamentally from cell separation during cytokinesis in animals, by leaving the coherence of the dividing protoplast basically intact. The resulting symplastic coherence appears more important for plant morphogenesis than histological structure; similar morphologies are realized on the basis of distinct tissue architectures in different plant taxa. The shape of a plant cell is determined by the shape its cell wall attains under multiaxial tensile stress. Consequently, the development of form in plants is achieved by a differential plastic deformation of the complex ECM in response to this multiaxial force (hydrostatic pressure). Current concepts of the regulation of these deformation processes are briefly evaluated.     

MECHANICAL BEHAVIOR OF PLANT TISSUES AS INFERRED FROM THE THEORY OF PRESSURIZED CELLULAR SOLIDS

The mechanical behavior of plant tissues and its dependency on tissue geometry and turgor pressure are analytically dealt with in terms of the theory of cellular solids. A cellular solid is any material whose matter is distributed in the form of beamlike struts or complete “cell” walls. Therefore, its relative density is less than one and typically less than 0.3. Relative density is the ratio of the density of the cellular solid to the density of its constitutive (“cell wall”) material. Relative density depends upon cell shape and the density of cell wall material. It largely influences the mechanical behavior of cellular solids. Additional important parameters to mechanical behavior are the elastic modulus of “cell walls” and the magnitude of internal “cell” pressure. Analyses indicate that two “stiffening” agents operate in natural cellular solids (plant tissues): 1) cell wall infrastructure and 2) the hydrostatic influence of the protoplasm within each cellular compartment. The elastic modulus measured from a living tissue sample is the consequence of both agents. Therefore, the mechanical properties of living tissues are dependent upon the magnitude of turgor pressure. High turgor pressure places cell walls into axial tension, reduces the magnitude of cell wall deformations under an applied stress, and hence increases the apparent elastic modulus of the tissue. In the absence of turgid protoplasts or in the case of dead tissues, the cell wall infrastructure will respond as a linear elastic, nonlinear elastic, or “densifying” material (under compression) dependent upon the magnitude of externally applied stress. Accordingly, it is proposed that no single tangent (elastic) modulus from a stress-strain curve of a plant tissue is sufficient to characterize the material properties of a sample. It is also suggested that when a modulus is calculated that it be referred to as the tissue composite modulus to distinguish it from the elastic modulus of a noncellular solid material.     

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.     

Wall extensibility: its nature, measurement and relationship to plant cell growth

Expansive growth of plant cells is controlled principally by processes that loosen the wall and enable it to expand irreversibly. The central role of wall relaxation for cell expansion is reviewed. The most common methods for assessing the extension properties of plant cell walls ( wall extensibility') are described, categorized and assessed critically. What emerges are three fundamentally different approaches which test growing cells for their ability (a) to enlarge at different values of turgor, (b) to induce wall relaxation, and (c) to deform elastically or plastically in response to an applied tensile force. Analogous methods with isolated walls are similarly reviewed. The results of these different assays are related to the nature of plant cell growth and pertinent biophysical theory. I argue that the extensibilities' measured by these assays are fundamentally different from one another and that some are more pertinent to growth than others.     

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)¹ (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself², and is amenable to high-throughput analyses³. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes⁴. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase^{5, 11, 12, 13}, xylan using an endo-β-(1-4)-xylanase ^6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase ⁸. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined ⁹.     

Cell wall water content has a direct effect on extensibility in growing hypocotyls of sunflower (Helianthus annuus L.)

It has been proposed that spacing between cellulose microfibrils within plant cell walls may be an important determinant of their mechanical properties. A consequence of this hypothesis is that the water content of cell walls may alter their extensibility and that low water potentials may directly reduce growth rates by reducing cell wall spacing. This paper describes a number of experiments in which the water potential of frozen and thawed growing hypocotyls of sunflower (Helianthus annuus L.) were altered using solutions of high molecular weight polyethylene glycol (PEG) or Dextran while their extension under constant stress was monitored using a creep extensiometer (frozen and thawed tissue was used to avoid confounding effects of turgor or active responses to the treatments). Clear reductions in extensibility were observed using both PEG and Dextran, with effects observed in hypocotyl segments treated with PEG 35 000 solutions with osmotic pressures of > or =0.21 MPa suggesting that the relatively mild stresses required to reduce water potentials of plants in vivo by 0.21 MPa may be sufficient to reduce growth rates via a direct effect on wall extensibility. It is noted, therefore, that the water binding capacity of plant cell walls may be of ecophysiological importance. Measurements of cell walls of sunflower hypocotyls using scanning electron microscopy confirmed that treatment of hypocotyls with PEG solutions reduced wall thickness, supporting the hypothesis that the spatial constraint of movement of cellulose microfibrils affects the mechanical properties of the cell wall.     

The relationship between xyloglucan endotransglycosylase and in-vitro cell wall extension in cucumber hypocotyls

It has been proposed that cell wall loosening during plant cell growth may be mediated by the endotransglycosylation of load-bearing polymers, specifically of xyloglucans, within the cell wall. A xyloglucan endotransglycosylase (XET) with such activity has recently been identified in several plant species. Two cell wall proteins capable of inducing the extension of plant cell walls have also recently been identified in cucumber hypocotyls. In this report we examine three questions: (1) Does XET induce the extension of isolated cell walls? (2) Do the extension-inducing proteins possess XET activity? (3) Is the activity of the extension-inducing proteins modulated by a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂)? We found that the soluble proteins from growing cucumber (cucumis sativum L.) hypocotyls contained high XET activity but did not induce wall extension. Highly purified wall-protein fractions from the same tissue had high extension-inducing activity but little or no XET activity. The XET activity was higher at pH 5.5 than at pH 4.5, while extension activity showed the opposite sensitivity to pH. Reconstituted wall extension was unaffected by the presence of a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂), an oligosaccharide previously shown to accelerate growth in pea stems and hypothesized to facilitate growth through an effect on XET-induced cell wall loosening. We conclude that XET activity alone is neither sufficient nor necessary for extension of isolated walls from cucumber hypocotyls.     

Lignin Biosynthesis Studies in Plant Tissue Cultures

Lignin, a phenolic polymer abundant in cell walls of certain cell types, has given challenges to scientists studying its structure or biosynthesis. In plants lignified tissues are distributed between other, non-lignified tissues. Characterization of native lignin in the cell wall has been difficult due to the highly cross-linked nature of the wall components. Model systems, like plant tissue cultures with tracheary element differentiation or extracellular lignin formation, have provided useful information related to lignin structure and several aspects of lignin formation. For example, many enzyme activities in the phenylpropanoid pathway have been first identified in tissue cultures. This review focuses on studies where the use of plant tissue cultures has been advantageous in structural and biosynthesis studies of lignin, and discusses the validity of tissue cultures as models for lignin biosynthesis.     

Mutations of the secondary cell wall

It has not been possible to isolate a number of crucial enzymes involved in plant cell wall synthesis. Recent progress in identifying some of these steps has been overcome by the isolation of mutants defective in various aspects of cell wall synthesis and the use of these mutants to identify the corresponding genes. Secondary cell walls offer numerous advantages for genetic analysis of plant cell walls. It is possible to recover very severe mutants since the plants remain viable. In addition, although variation in secondary cell wall composition occurs between different species and between different cell types, the composition of the walls is relatively simple compared to primary cell walls. Despite these advantages, relatively few secondary cell wall mutations have been described to date. The only secondary cell wall mutations characterised to date, in which the basis of the abnormality is known, have defects in either the control of secondary cell wall deposition or secondary cell wall cellulose or lignin biosynthesis. These mutants have, however, provided essential information on secondary cell wall biosynthesis.