Viscoelastic versus plastic cell wall extensibility in growing seedling organs: a contribution to avoid some misconceptions

In a recent publication (Kutschera, 1996), it was reported thatthe cell walls of growing rye coleoptiles exhibit irreversible(plastic) extensibility in a rheological extension test. Basicallysimilar measurements with cell walls of maize coleoptiles hadpreviously shown that the apparent plastic extensibility determinedin this material is in reality due to the slowly reversible(viscoelastic) extensibility of the walls. A recent reinvestigationof this discrepancy showed that rye coleoptile walls also behaveas a perfectly viscoelastic material if precautions are takento prevent measuring artefacts. Similar results were obtainedwith cell walls from the growing zone of various other seedlingorgans (maize mesocotyl, maize root, cucumber hypocotyl). Itis concluded that plastic extensibility has not yet been convincinglydemonstrated by rheological tests that determine the intrinsicmaterial properties of cell walls. Reported changes in mechanicalmaterial properties of cell walls produced by growth-controllingfactors such as auxin or light may generally be attributed tochanges in viscoelasticity which are not directly related tothe chemo-rheological processes controlling wall extension ofgrowing cells. Key words: Cell wall extensibility, extension growth, plastic cell wall extensibility, viscoelastic cell wall extensibility     

Calcium and the cell wall

Abstract. From this brief review it appears that the interactions between calcium ions and cell walls play a key role in plant physiology. Calcium ions are involved in many mechnisms: for example, stabilization of cell wall structures, acidic growth, ion exchange properties, control of the activities of wall enzymes. All these properties originate from the tight binding of calcium ions to the pectins present in the cell walls. The factor most important for controlling wall behaviour is the density of non-diffusible charges and, due to its high affinity, calcium can significantly affect this factor. We also discuss the theoretical ion exchange models in relation to the specific role of calcium ions.     

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.     

Interaction between wall deposition and cell elongation in dark-grown hypocotyl cells in Arabidopsis

A central problem in plant biology is how cell expansion is coordinated with wall synthesis. We have studied growth and wall deposition in epidermal cells of dark-grown Arabidopsis hypocotyls. Cells elongated in a biphasic pattern, slowly first and rapidly thereafter. The growth acceleration was initiated at the hypocotyl base and propagated acropetally. Using transmission and scanning electron microscopy, we analyzed walls in slowly and rapidly growing cells in 4-d-old dark-grown seedlings. We observed thick walls in slowly growing cells and thin walls in rapidly growing cells, which indicates that the rate of cell wall synthesis was not coupled to the cell elongation rate. The thick walls showed a polylamellated architecture, whereas polysaccharides in thin walls were axially oriented. Interestingly, innermost cellulose microfibrils were transversely oriented in both slowly and rapidly growing cells. This suggested that transversely deposited microfibrils reoriented in deeper layers of the expanding wall. No growth acceleration, only slow growth, was observed in the cellulose synthase mutant cesA6(prc1-1) or in seedlings, which had been treated with the cellulose synthesis inhibitor isoxaben. In these seedlings, innermost microfibrils were transversely oriented and not randomized as has been reported for other cellulose-deficient mutants or following treatment with dichlorobenzonitrile. Interestingly, isoxaben treatment after the initiation of the growth acceleration in the hypocotyl did not affect subsequent cell elongation. Together, these results show that rapid cell elongation, which involves extensive remodeling of the cell wall polymer network, depends on normal cellulose deposition during the slow growth phase.     

Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth

Hydroxyl radicals (*OH), produced in the cell wall, are capable of cleaving wall polymers and can thus mediate cell wall loosening and extension growth. It has recently been proposed that the biochemical mechanism responsible for *OH generation in the cell walls of growing plant organs represents an enzymatic reaction catalyzed by apoplastic peroxidase (POD). This hypothesis was investigated by supplying cell walls of maize ( Zea mays L.) coleoptiles and sunflower ( Helianthus annuus L.) hypocotyls with external NADH, an artificial substrate known to cause *OH generation by POD in vitro. The effects of NADH on wall loosening, growth, and *OH production in vivo were determined. NADH mediates cell wall extension in vitro and in vivo in an H2O2-dependent reaction that shows the characteristic features of POD. NADH-mediated production of *OH in vivo was demonstrated in maize coleoptiles using electron paramagnetic resonance spectroscopy in combination with a specific spin-trapping reaction. Kinetic properties and inhibitor/activator sensitivities of the *OH-producing reaction in the cell walls of coleoptiles resembled the properties of horseradish POD. Apoplastic consumption of external NADH by living coleoptiles can be traced back to the superimposed action of two enzymatic reactions, a KCN-sensitive reaction mediated by POD operating in the *OH-forming mode, and a KCN-insensitive reaction with the kinetic properties of a superoxide-producing plasma-membrane NADH oxidase the activity of which can be promoted by auxin. Under natural conditions, i.e. in the absence of external NADH, this enzyme may provide superoxide (O2*-) (and H2O2 utilized by POD for) *OH production in the cell wall.     

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.     

Properties of cellulose/pectins composites: Implication for structural and mechanical properties of cell wall

The primary cell wall of dicotyledonous plants can be considered as a concentrated polymer assembly, containing in particular polysaccharides among which cellulose and pectins are known to be the major components. In order to understand and control the textural quality of plant-derived foods, it is highly important to elucidate the rheological and microstructural properties of these components, individually and in mixture, in order to define their implication for structural and mechanical properties of primary plant cell wall. In this study, the rheological and microstructural properties of model systems composed of sugar-beet microfibrillated cellulose and HM pectins from various sources, with varied degrees of methylation and containing different amounts of neutral sugar side chains, were investigated. The influence of the presence of calcium and/or sodium ions and the biopolymer concentrations on the properties of the mixed systems were also studied. The characterizations of the mixed system, considered as a simplified model of primary plant cell wall, showed that whatever the structural characteristics of the pectins, the ionic conditions of the medium and the biopolymer concentrations, the gelation of the composite was mainly controlled by cellulose. Thus, the cellulose network would be the principal component governing the mechanical properties of the cell walls. However, the neutral sugar side chains of the pectins seem to play a part in the interactions with cellulose, as shown by the interesting viscoelastic properties of cellulose/apple HM pectins systems. The rigidity of cellulose/pectins composite was strongly influenced by the structural characteristics of pectins. The particular properties of primary plant cell walls would thus result from the solid viscoelastic properties of cellulose, its interactions with pectins according to their structural characteristics (implication of the neutral sugar side chains and the specific potential calcic interactions) and of the distribution of the components in separate phases.     

Loss of stability-a new model for stress relaxation in plant cell walls

This study addresses the mechanism of wall stress relaxation in growing plant cells. The current viscoelastic model of cell wall relaxation, which dates from the work of Preston, Cleland, Lockhart, and others in the 1960s, has serious shortcomings. It has been shown however that the theory of loss of stability (LOS) can be applied to materials in tension, leading to the conclusion that the relaxation of stresses in the walls of any pressure vessel is rigorously modeled using LOS. We propose that LOS also provides a more appropriate and versatile model of stress relaxation in growing plant cells. We argue that when treated as a manifestation of LOS, the regulation of cell turgor has a rigorous and demonstrable basis in the geometrical and physical properties of the cell wall and the cell's ability to import water. Thus plant cell growth can be regarded as an inherently self-limiting process, tunable by biochemical or structural means. Lastly, despite the current limitations of our model, we apply direct measurement of elastic modulus, wall thickness and cell radius obtained from cylindrical Chara corallina cells to generate an initial calculation of critical pressures in a hypothetical spherical cell with the same material properties.     

Guard cell wall: immunocytochemical detection of polysaccharide components

The composition of guard cell walls in sugar beet leaves (Beta vulgaris L.) was studied by using histochemical staining and immunocytochemical detection of cell wall antigens. The findings were compared with those in the walls of epidermal and mesophyll cells. Probing of leaf sections with monoclonal antibodies against pectins, terminal fucosyl residues linked alpha-(1-->2) to galactose, beta-(1-->3)-glucans and arabinogalactan-proteins revealed several specific features of guard cells. Pectic epitopes recognized by JIM7 were homogeneously distributed in the wall, whereas pectins recognized by JIM5 were not found in the walls themselves, but were abundant in the cuticular layer. Large amounts of molecules bearing terminal fucose were located predominantly in ventral and lateral guard cell walls. Much smaller amounts were detected in dorsal walls of these cells, as well as in the walls of pavement and mesophyll cells. Conspicuous accumulation of these compounds was observed in the vicinity of the guard cell plasmalemma, whereas labelling was scarce in the areas of the wall adjacent to the cell surface. The presence of callose clearly marked the ventral wall between the recently formed, very young guard cells. Callose also appeared in some mature walls, where it was seen as punctate deposits that probably reflected a specific physiological state of the guard cells. Large amounts of arabinogalactan-proteins were deposited within the cuticle, and smaller amounts of these proteoglycans were also detected in other tissues of the leaf. The histochemical and immunocytochemical structure of the guard cell wall is discussed in the light of its multiple functions, most of which involve changes in cell size and shape.     

The plant cell pressure probe

The pressure probe is a micro manometer for the simultaneous direct recording and manipulation of plant cell hydrostatic pressure. It is used to map in space and time the turgor pressures of individual cells within tissues and organs of intact plants. This is used to study the hydraulic architecture of tissues, tissue movement and the responses of tissues to water stress. The approach can be augmented by simultaneous measurement of individual cell osmotic pressure. This permits the hydraulic driving forces across selectively permeable membranes and walls to be assessed fully. By manipulating manually the pressure, cell wall elasticity and its properties can also be mapped. Under some conditions this can be extended to plastic behaviour.     

Viscoelastic properties of cell walls of single living plant cells determined by dynamic nanoindentation

Plant development results from controlled cell divisions, structural modifications, and reorganizations of the cell wall. Thereby, regulation of cell wall behaviour takes place at multiple length scales involving compositional and architectural aspects in addition to various developmental and/or environmental factors. The physical properties of the primary wall are largely determined by the nature of the complex polymer network, which exhibits time-dependent behaviour representative of viscoelastic materials. Here, a dynamic nanoindentation technique is used to measure the time-dependent response and the viscoelastic behaviour of the cell wall in single living cells at a micron or sub-micron scale. With this approach, significant changes in storage (stiffness) and loss (loss of energy) moduli are captured among the tested cells. The results reveal hitherto unknown differences in the viscoelastic parameters of the walls of same-age similarly positioned cells of the Arabidopsis ecotypes (Col 0 and Ws 2). The technique is also shown to be sensitive enough to detect changes in cell wall properties in cells deficient in the activity of the chromatin modifier ATX1. Extensive computational modelling of the experimental measurements (i.e. modelling the cell as a viscoelastic pressure vessel) is used to analyse the influence of the wall thickness, as well as the turgor pressure, at the positions of our measurements. By combining the nanoDMA technique with finite element simulations quantifiable measurements of the viscoelastic properties of plant cell walls are achieved. Such techniques are expected to find broader applications in quantifying the influence of genetic, biological, and environmental factors on the nanoscale mechanical properties of the cell wall.     

Two endogenous proteins that induce cell wall extension in plants.

Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.     

Pattern and process of wall formation in developing endosperm

Endosperm is emerging as a system for investigating the genetic control of wall placement and deposition in plant development. Development of endosperm progresses in distinct stages from a wall-less syncytial stage to a cellular stage that is entirely typical of plant meristems where the division plane is predicted by a preprophase band of microtubules (PPB) and cytokinesis is completed by formation of a cell plate in association with a phragmoplast. Four developmentally different types of walls, each associated with a different microtubule system, are sequentially produced: (1) free growing walls deposited in the absence of mitosis and phragmoplasts; (2) walls guided by cytoplasmic phragmoplasts formed adventitiously in the absence of mitosis; (3) walls formed by interzonal phragmoplasts in a cell cycle that lacks PPBs; and (4) wall deposition driven by interzonal phragmoplasts in a cycle that includes PPBs. We are using methods of differential screening to isolate cDNA clones corresponding in temporal and spatial pattern to the types of wall development, and are studying mutants for clues to the genetic controls of wall development.     

Auxin-dependent cell wall depositions in the epidermal periplasmic space of graviresponding nodes of Tradescantia fluminensis

Differential growth of the nodal regions of graviresponding Tradescantia fluminensis (Wandering Jew) was analysed with special respect to the extension-restricting epidermal cells of the opposite growing and growth-inhibited organ flanks. Gravicurvature of horizontally gravistimulated isolated nodes depends on auxin (indolyl-3-acetic acid, IAA) and shows a node-specific profile in which the third node below the tip showed the greatest response. Exogenously supplied gibberellic acid induced no gravitropic growth. Vertically oriented isolated nodes supplied with exogenous IAA showed, on an electron microscopical level, conspicuous membrane invaginations with adjacent wall depositions restricted to the outer tangential epidermal cell walls. Their number was more than doubled by exogenously supplied Ca2+, which inhibited IAA-induced growth. No such changes could be detected in water-incubated segments or inner tissues of IAA-supplied segments. Gravistimulated differential growth of nodes of intact shoots and of nodal segments was characterized by changes similar to the ones induced by exogenous IAA, with greatly increased numbers of wall depositions within the epidermal cells of the growth-inhibited upper organ flank. Similar to the gravistimulated wall depositions, an asymmetric distribution pattern of Ca2+ was detected in the epidermal cell walls employing x-ray energy spectrum analysis (EDX). The results indicate that growth of nodes of Tradescantia fluminensis is regulated via IAA-induced secretion and subsequent infiltration of wall components enabling wall extension. The data support the hypothesis that temporary differential growth during gravicurvature of Tradescantia fluminensis is mediated by the antagonistic effect of Ca(2+)-ions on the infiltration of IAA-induced wall-loosening components into the outer, extension-restricting epidermal walls thereby inhibiting growth.     

Endo-xyloglucan transferase,a new class of transferase involved in cell wall construction

Cell shape in plants is constrained by cell walls, which are thick yet dynamic structures composed of crystalline cellulose microfibrils and matrix polymers. Xyloglucans are the principal component of the matrix polymers and bind tightly to the surface of cellulose microfibrils and thereby cross-link them to form an interwoven xyloglucan-cellulose network structure. Thus, cleavage and reconnection of the cross-links between xyloglucan molecules are required for the rearrangement of the cell wall architecture, the process essential for both cell wall expansion and the wall deposition occurring during cell growth and differentiation. Endoxyloglucan transferase (EXT) is a newly identified class of transferase that catalyzes molecular grafting between xyloglucan molecules. This enzyme catalyzes both endo-type splitting of a xyloglucan molecule and reconnection of a newly generated reducing terminus of the xyloglucan to the non-reducing terminus of another xyloglucan molecule, thereby mediating molecular grafting between xyloglucan cross-links in plant cell walls. Molecular cloning and sequencing of EXT-cDNAs derived from five different plant species includingA. thaliana andV. angularis has revealed that the amino acid sequence of the mature protein is extensively conserved in the five different plant species, indicating that EXT protein is ubiquitous among higher plants. This structural study has also disclosed the presence of a group of xyloglucan related proteins (XRPs) with transferase activity in higher plants. Current data strongly suggest that these proteins are involved in a wide spectrum of physiological activities including cell wall expansion and deposition in growing cell walls. Recipient of the Botanical Sociaty Award of Young Scientists, 1993.     

Polygalacturonase-inhibiting protein is a structural component of plant cell wall

It is generally believed that plants "evolved a strategy of defending themselves from a phytopathogen attack" during evolution. This metaphor is used frequently, but it does not facilitate understanding of the mechanisms providing plant resistance to the invasion of foreign organisms and to other unfavorable external factors, as well as the role of these mechanisms in plant growth and development. Information on processes involving one of the plant resistance factors--polygalacturonase-inhibiting protein (PGIP)--is considered in this review. The data presented here indicate that PGIP, being an extracellular leucine-rich repeat-containing protein, performs important functions in the structure of plant cell wall. Amino acid residues participating in PGIP binding to homogalacturonan in the cell wall have been determined. The degree of methylation and the mode of distribution of homogalacturonan methyl groups are responsible for the formation of a complex structure, which perhaps determines the specificity of PGIP binding to pectin. PGIP is apparently one of the components of plant cell wall determining some of its mechanical properties; it is involved in biochemical processes related to growth, expansion, and maceration, and it influences plant morphology. Polygalacturonase (PG) is present within practically all plant tissues, but the manifestation of its activity varies significantly depending on physiological conditions in the tissue. Apparently, the regulation of PG functioning in apoplast significantly affects the development of processes associated with the modification of the structure of plant cell wall. PGIP can regulate PG activity through binding to homogalacturonan. The genetically determined structure of PGIP in plants determines the mode of its interaction with an invader and perhaps is one of the factors responsible for the set of pathogens causing diseases in a given plant species.