JXB Advance Access originally published online on August 1, 2005
Journal of Experimental Botany 2005 56(419):2275-2285; doi:10.1093/jxb/eri247
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OPINION PAPER |
How do cell walls regulate plant growth?
School of Biosciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK
* Fax: +44 (0)20 7911 5800, ext 5087. E-mail: thompss{at}wmin.ac.uk
Received 24 March 2005; Accepted 9 June 2005
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Abstract |
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The cell wall of growing plant tissues has frequently been interpreted in terms of inextensible cellulose microfibrils ‘tethered’ by hemicellulose polymers attached to the microfibril surface by hydrogen bonds, with growth occurring when tethers are broken or ‘peeled’ off the microfibril surface by expansins. This has sometimes been described as the ‘sticky network’ model. In this paper, a number of theoretical difficulties with this model, and discrepancies between predicted behaviour and observations by a number of researchers, are noted. (i) Predictions of cell wall moduli, based upon the sticky network model, suggest that the cell wall should be much weaker than is observed. (ii) The maximum hydrogen bond energy between tethers and microfibrils is less than the work done in expansion and therefore breakage of such hydrogen bonds is unlikely to limit growth. (iii) Composites of bacterial cellulose with xyloglucan are weaker than pellicles of pure cellulose so that it seems unlikely that hemicelluloses bind the microfibrils together. (iv) Calcium chelators promote creep of plant material in a similar way to expansins. (v) Reduced relative ‘permittivities’ inhibit the contraction of cell wall material when an applied stress is decreased. Revisions of the sticky network model that might address these issues are considered, as are alternatives including a model of cell wall biophysics in which cell wall polymers act as ‘scaffolds’ to regulate the space available for microfibril movement. Experiments that support the latter hypothesis, by demonstrating that reducing cell wall free volume decreases extensibility, are briefly described.
Key words: Cell wall, expansins, plant growth, rheology
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Introduction |
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TopAbstractIntroductionEvidence inconsistent with the...Alternatives to the sticky...ConclusionsReferences |
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Many plant structures derive their strength and stability from the interaction between relatively high internal pressures (typically from 0.3–1.5 MPa) and a rigid cell wall, which is usually maintained in a state of tension by the cell turgor pressure. This arrangement presents a problem, in that growing cells need to be able to modify the cell wall dimensions without allowing its resilience to be compromised. It has become generally accepted that this is achieved by a combination of slow stretching of material that has already been laid down and the deposition of new material to maintain wall integrity. This model suggests that the mechanical properties of the primary cell wall are of substantial importance in regulating plant growth. This paper reappraises the current dominant model of how cell wall behaviour arises from the interaction of its individual components and how growth occurs.
Examination of cell wall structure reveals an array of cellulose microfibrils with surfaces coated with hemicelluloses, attached to the microfibril surface by hydrogen bonds (McCann et al., 1990
). This network is interpenetrated by pectins. Many recent models of plant growth have assumed that, in primary cell walls, hemicelluloses bridging the gaps between microfibrils act as load bearing cross-links or tethers, and that growth occurs when these bridging polymers are broken or removed from the microfibril surface (Passioura and Fry, 1992). This has been to referred to as the ‘sticky network’ model by Cosgrove (2000).
This scheme appears intuitively attractive and provides obvious explanations of why plant growth often seems to cease if turgor pressure falls below a certain value (commonly known as the ‘yield threshold’) in terms of minimum force required to break hydrogen bonds and extensibility in terms of rate of breakage (Passioura and Fry, 1992). The model also suggests clear roles for a number of enzymes that appear to be associated with growth such as xyloglucan-endotransglycosylase (XET) (Fry et al., 1992; Palmer and Davies, 1996) and endoglucanases (Loopstra et al., 1998; Park et al., 2003) in breaking hemicellulose tethers, and for expansins, which are thought to break hydrogen bonds between cellulose microfibrils and hemicelluloses (McQueen-Mason and Cosgrove, 1994).
Presumably, as each tether is stretched, elastic energy would be stored in the tether (in this case as a reduction in conformational entropy) until it balanced the energy binding the next segment of the tether to the microfibril, at which point part of the tether would be released from the microfibril surface leading to permanent deformation of the cell wall and restoring chain conformational entropy (Sperling, 1997).
However, this interpretation would require that wall stress is transmitted through both the hemicellulose and cellulose components of the wall so that both bear the total load (i.e. that the cellulose–hemicellulose network approximates an ‘isostress’ system). The wall modulus can be calculated for an isostress model using Takayanagi models from the equation below (Sperling, 1997).
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C and HC are the volume fractions of cellulose and hemicellulose, respectively, and EC and EHC are their respective moduli. For this (as well as more complex arrangements in which the tethers are load bearing) the wall modulus should tend towards that of the hemicellulose. Moduli for hemicelluloses are typically very much lower than those of cellulose measured under similar conditions, but cell walls have moduli of the same order of magnitude as cellulose (Whitney et al., 1999). This suggests that the cell wall, in fact, behaves much more like an isostrain system, in which the components are constrained to the same length and the stress is distributed between the components in proportion to volume fraction and modulus. If the cell wall is an isostrain system, the cellulose should bear the vast majority of the wall stress. Therefore it seems appropriate to re-examine the sticky network model with particular emphasis on evidence that may contradict the model.
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Evidence inconsistent with the ‘sticky network’ model |
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TopAbstractIntroductionEvidence inconsistent with the...Alternatives to the sticky...ConclusionsReferences |
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Work done during cell wall extension is greater than the total hydrogen bond energy of interactions between hemicellulose and cellulose microfibrils and therefore these interactions cannot limit growth rates
Plant cells typically have turgor pressures of 0.3–1.5 MPa. Cell walls of growing tissues are relatively thin, and so stresses in the cell wall are reported to be 10–100 MPa (Cosgrove, 1997
). Work (W) done in an extending wall can be calculated from the stress (
), area (A), and change in length (
L) and is given by:
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The ‘true strain’, given by ln(Lt/Lo), would give a more accurate estimate, but the differences are not substantial (only reducing maximum work by 15%).
Frozen and thawed cell walls from growing tissues can extend by more than 40% (Cleland et al., 1987). This would take 4–40 MJ m–3 work for unit area of cell wall material in vivo.
The density of hydrogen bonds was calculated from the surface area of the cellulose microfibrils, and used to calculate the total bond energy as follows.
- (i) A unit cell of cellulose of two glucose monomers measures 1.03x0.79 nm (Brett and Waldron, 1990) and provides sites for four hydrogen bonds (Finkenstadt et al., 1995).
- (ii) Hydrogen bond energies are about 20 kJ mol–1 in vacuo but typically only 6.5 kJ mol–1 in an aqueous environment (Miller, 1966
; Sheu et al., 2003).
- (iii) Cellulose microfibrils have diameters of 5–10 nm (McCann et al., 1990
) giving an area:volume ratio of 4–8x108 m2 m–3. NMR and X-ray diffraction measurements of microfibril size in celery petioles suggested that diameters were only 2–3 nm in diameter (Thimm et al., 2002), but these methods may only detect crystalline portions of the microfibrils and atomic force microscopy of hydrated walls of the same tissue indicated microfibril diameters to be 6–25 nm (Thimm et al., 2000). The cell wall is 65% water and 30% of the remainder is cellulose, which therefore makes up about 10% of the wall volume.
- (ii) Hydrogen bond energies are about 20 kJ mol–1 in vacuo but typically only 6.5 kJ mol–1 in an aqueous environment (Miller, 1966
).
Some additional hydrogen bond energy could result from ‘recycling’ of binding sites, but a significant degree of recycling places a limit upon the maximum length of each stretch of hemicellulose binding to the microfibril surface. This is because, for recycling to make a substantial contribution, relatively limited extensions must lead to ‘turnover’ of a high proportion of the total binding sites, requiring that most of the residues attaching each tether must be removed so that the area they occupy on the microfibril surface can be reoccupied by new tethers. Cellulose microfibrils are separated by 20–40 nm (McCann et al., 1990) and so 4–8 glucose residues per tether should be removed from the microfibril surface for each 10% length increase (from unit cell dimensions). Recycling of all binding sites during extension by 20% would therefore require each tether to be bound to the microfibril surface by stretches of about 8–16 residues, limiting tether separation on the microfibril to <10 nm, much closer than the spacing between tethers visible in electron micrographs (McCann et al., 1990). New tethers utilizing released binding sites are also equally likely to form laterally or radially, or form non-tethering loops leading to progressive weakening of the wall, something that is not generally observed.
Entropic contributions were not considered in the above calculations, but would fall into two categories: (i) conformational entropy of the tethering chain; (ii) entropic differences between segments of hemicellulose bound to the cellulose surface and unbound segments.
As noted in the Introduction, the former is likely to decrease in each tether until it generates a free energy similar in value to the binding energy and then to increase again by a similar value and can therefore probably be ignored. The entropic change in releasing hemicellulose segments could hypothetically be negative, most plausibly if either the microfibril surface or hemicelluloses had an ‘ordering’ effect on surrounding water molecules and in which case binding to the microfibril would be favoured both energetically and entropically, but it seems substantially more likely that release of segments of tether would involve an entropy increase, and therefore increase the gap between hydrogen bonding energy and work done in extension.
The analysis above is based upon a number of assumptions. The proportion of length change that is due to elastic extension has been ignored. This can be justified as the work done in elastic extension in an isostress model must be balanced by hydrogen bonds at the microfibril surface, so that even if not all work done during extension is involved in breaking bonds, the remaining unbroken hydrogen bonds must have a total binding energy greater than the total elastic energy. However, there are three other areas that seem to require further consideration as they bear upon the conclusions that can be drawn from the calculations and the sticky network model itself.
- (i) It has been assumed that interaction behaves as if it is in an aqueous environment. If binding takes place in a local environment of lower relative permittivity, hydrogen bond strengths could approach 20 kJ mol–1 and the density of hydrogen bond energy in the cell wall would be 7–13 MJ m–3, plausibly sufficient to stabilize the wall. This type of behaviour occurring in polysaccharides has not been heard of by the author, but examples of such protected environments from protein chemistry are common. This interpretation also suggests a possible mechanism for expansin activity, as hydrogen bonds between the tethers and microfibrils could be dramatically weakened by modification of the bonding environment. In addition, pectin concentrations in the wall are extremely high (Darley et al., 2001) and might reduce water activity and, therefore, relative permittivity.
- (ii) Much cellulose at the microfibril surface is thought to be ‘amorphous’ (Rose and Bennett, 1999
) and it is possible that this provides a greater area for binding than an ordered surface. In addition, hemicelluloses may be woven into the microfibrils (Pauly et al., 1999). This could increase the area of interaction and perhaps reduce the relative permittivity in which binding occurs. The observation that expansins bind preferentially to areas of amorphous cellulose is consistent with this.
- (iii) An unexpected prediction of the modelling of total hydrogen bond energy presented above is that for the same total quantity of cellulose, smaller microfibrils should lead to a stronger wall in the sticky network model because of the greater surface area for tether formation. Were the microfibril diameter smaller than assumed (for example the 2–3 nm diameter suggested by NMR and X-ray diffraction; Thimm et al., 2000
) or were the microfibrils flattened or ribbon shaped, the additional area for binding would increase the total hydrogen bond energy.
- (ii) Much cellulose at the microfibril surface is thought to be ‘amorphous’ (Rose and Bennett, 1999
Composites of cellulose with hemicelluloses are weaker than pure cellulose and of comparable strength to composites with pectin
Several recent publications have described the properties of composites of bacterial cellulose with plant cell wall polysaccharides (Chanliaud and Gidley, 1999; Whitney et al., 1999). All of the composites extended more than pure cellulose at equivalent stress (i.e. had a reduced strain modulus) and exhibited a reduced break stress.
Although pectins are not thought to associate with the composite microfibrils in the same way as hemicelluloses, composites with pectins behaved in broadly the same way as composites with xyloglucans and glucomannans, but it was found that increased extension after treatment with expansins only occurred in composites with xyloglucan, suggesting that xyloglucans are specifically related to the roles of some expansins (Whitney et al., 2000).
The effects of extraction or enzymic modification of the non-cellulosic components of the composites was also surprising. Properties of composites with pectin did not converge with those of pure cellulose after extraction of the pectin (Chanliaud and Gidley, 1999), but remained comparable to those of the composites prior to extraction. Furthermore, treatment of composites with xyloglucan with xyloglucanase, endo-glucanase or with xyloglucan endo transglycosylase in the presence of xyloglucan oligosaccharides, all treatments expected to break tethers, increased strain modulus and decreased creep (Chanliaud et al., 2004).
Comparison of creep of hydrated Acetobacter xylinus cellulose with creep of plant material also suggests that, although it is much less extensible than cell walls from growing tissue, the rheological behaviour of pure bacterial cellulose exhibits the same type of rheological elements as the plant cell wall, whereas simpler behaviour might be expected if more components were load bearing in the plant cell wall. Creep of bacterial cellulose was measured as previously described for tomato fruit epidermis (Thompson, 2001). Various rheological models were compared with the data. The model found to correspond most closely was composed of a log–time function, two Kelvin elements, and a viscous flow element. This is identical to the model which was found to provide the best fit to extension of tomato fruit epidermis (Thompson, 2001), pea leaves (Stam-Bolink, 2003), tomato leaf rachis, wheat seed coats, and sunflower hypocotyls (DS Thompson, unpublished observations). Figure 1 shows creep data for Acetobacter xylinus cellulose, together with a curve fitted to the data using this model, as described in Thompson (2001). The model parameters for Acetobacter cellulose and a strip of tomato fruit epidermis are compared in Table 1. This illustrates that although extension of bacterial cellulose was much more limited than plant material and that, consequently, the constant for the log–time function (Ncreep) and flow rates (f1, f2, and f3) were substantially different, the retardation times of the two Kelvin elements (
1 and 2) corresponded reasonably closely with those of the plant material (also note the retardation times for sunflower hypocotyls in Table 2). Retardation and relaxation times are often interpreted as being specific to particular types of process, with shorter times being associated with smaller scale events such as rotation of polymer segments relative to one another and longer times associated with larger scale events such as relative movement of polymer chains (Matsuoka, 1992). The similarity of retardation times can, therefore, be interpreted as suggesting that, although the degree to which each manifests is different in the two materials, extension in the pure cellulose composites and the cell wall involves fundamentally similar processes.
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Calcium chelators promote creep of plant tissue
In an early paper that defined expansin behaviour before the proteins had been specifically identified (Cosgrove, 1989
) it was noted that the calcium chelator EDTA had similar effects to acidic pH. This behaviour was attributed to the lack of specificity of EDTA, as it was known that other metal ions, such as those of copper (which are bound by EDTA), inhibit creep at acid pH. However, it is possible that increased extension was a direct effect of EDTA on the crosslinking of the pectin network.
This experiment was repeated using BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid), a chelator that binds calcium much more strongly than magnesium ions or protons (Tsien, 1980). The creep of tomato epidermal strips in the presence of 1 mM CaCl2, 5 mM BAPTA, and 5 mM BAPTA with 6 mM CaCl2 (i.e. BAPTA in the presence of approximately 1 mM Ca2+) are compared in Fig. 2. Increased creep in the presence of BAPTA demonstrates that the effect is not due to Mg2+. More importantly, because BAPTA binds many heavier ions (such as Cu2+) much more strongly than Ca2+ (Loukin and Kung, 1995), the reduction of creep by the addition of a sufficiently high concentration of Ca2+ to give a free Ca2+ concentration of approximately 1 mM, strongly suggests that the removal of ions of heavier metals is not responsible for increased creep in the presence of chelators, as the addition of Ca2+ would not release substantial quantities of them (for example, from the relative Kd values, the concentration of Cu2+ released by the addition of 6 mM CaCl2 would be approximately 6 nM). Furthermore, the effect of calcium chelators appears to be independent of expansin action, as the effect can be observed at neutral pH as well as in walls that have been boiled in order to inactivate expansins.
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The effect of calcium chelators upon the parameters used to model creep of tomato fruit epidermis, using the model described above, is also extremely similar to that of reduced pH (although with a slightly greater effect on parameters that predominate during early creep). It was found that under most circumstances the effects of reduced pH (and therefore presumably expansin activity) and calcium chelators were not additive, although some additional effect of pH occurred in material from the fastest growing tissue.
These results suggest either that the pectins in the cell wall are also of importance in determining growth rate or that expansin activity involves an interaction with calcium ions or with pectins (it should be noted that there is no evidence for or against expansins exhibiting either type of interaction, but they are included as logical possibilities).
Reduced relative permittivities inhibit both extension and contraction of cell wall material, whereas the sticky network model would predict only inhibition of extension
Edelmann (1995) investigated the effect of non-covalent bonds on cell wall behaviour by examining the effect of non-polar solvents on cell wall properties. The rationale was that hydrogen bonds would be strengthened in an environment with reduced relative permittivity and that this would modify wall properties if hydrogen bonds or other non-covalent interactions were important in determining its behaviour.
It was found that after elimination of turgor pressure, plant tissues in methanol did not contract until transferred to water, which was interpreted as indicating that increased hydrogen bond energy in methanol had fixed the tissue in a state of elastic extension (the relative permittivities of water and methanol are, respectively, 78.5 and 32.7).
Creep of cell walls of tomato fruit epidermis in 100% ethanol confirms that cell walls do exhibit substantially reduced creep, as illustrated in Fig. 3a. When stress was increased, extension was much less than in strips incubated in aqueous buffer, but substantial extension occurred when ethanol was replaced with MES buffer. A virtually symmetrical effect was observed when stress was removed (Fig. 3b). Initial contraction of the strips in ethanol was limited, but a considerable degree of further shrinkage occurred when the bathing medium was changed.
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The inhibition of extension is consistent with the sticky network model, but inhibition of contraction is much harder to explain in these terms because contraction should involve shortening of tethers due to elastic contraction (as a large proportion of stored elastic energy would be expected to reside in the xyloglucan tethers if they were load bearing). It is hard to see how strengthening of hydrogen bonding would prevent this and it might even be expected to increase the proportion of stored elastic energy released in tether shortening as opposed to the alternate energy release mechanism of ‘peeling’ off the microfibril surface. Although it is possible that methanol and ethanol have specific effects upon the wall components that are independent of relative permittivity, if their effect is attributable to strengthening of non-covalent interactions, it is inconsistent with the sticky network model.
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Alternatives to the sticky network model |
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TopAbstractIntroductionEvidence inconsistent with the...Alternatives to the sticky...ConclusionsReferences |
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If it is accepted that the sticky network model, as currently formulated, is not entirely satisfactory, revisions might include re-examination of the role of hemicellulose incorporation into the microfibril structure or consideration of specific and unusual energetic behaviour at the microfibril surface. Alternatively, the cell wall could be interpreted in different terms. A number of alternatives to the sticky network model are considered below.
The multi-coat model
Proposed by Talbott and Ray (1992), this model is roughly contemporaneous with the sticky network model. In this interpretation of the wall, hemicelluloses coating the microfibrils are not seen as direct bridges or tethers between microfibrils, but instead interact with other hemicelluloses and pectins in the space between them and which, in turn, interact with hemicelluloses coating other microfibrils to form indirect links.
This model is certainly consistent with the effects of calcium chelators (which could act by weakening the cross-linking in the intermediate pectin layers) and the effect of non-polar solvents (as the intermediate interactions could resist extension or contraction). However, if the calculation of cell wall hydrogen bond energy at the microfibril surface is sound, it appears that the main site of resistance to extension would have to be in the matrix between the microfibrils. This would be surprising, as it appears that expansins have a specific affinity for the microfibrils. In addition, this interpretation would require that the cell wall be an isostress system in the same way as the sticky network model and the various non-cellulosic components of the cell wall are not particularly robust in isolation. Nonetheless, the extremely high concentrations of pectin in the cell wall may behave in unexpected ways and expansins may be able to act on non-covalent interactions between polysaccharides other than xyloglucan and cellulose (again this possibility is included for logical completeness rather than a basis in published evidence).
Rejection of the Lockhart paradigm
The hypothesis that plant growth results from the interaction between cell wall stress and cell wall mechanical properties was initially proposed by Lockhart and has been broadly accepted since that time (Lockhart, 1965), but some papers have argued that extension in vivo involves processes normally requiring metabolic activity and that mechanical properties of cell walls in vitro are not directly related to growth, implicitly suggesting rejection of this paradigm (Ding and Schopfer, 1997). An element of this argument is the claim that measurements of ‘plastic deformation’ of plant tissue have, in fact, been a misinterpretation of hysteresis and that if loads are reduced to zero and enough time is allowed for recovery, plant cell walls do not exhibit permanent deformation (Hohl and Schopfer, 1992; Nolte and Schopfer, 1997). Instead it has been proposed that extension is a result of active redox processes (Schopfer, 2001).
While it is certainly the case that much short-term creep is usually reversible and that it seems likely that the ‘plastic extension’ detected by Instron measurements probably reflects the previous history of the material as well as its mechanical properties (Cleland, 1984), these criticisms do not seem to be applicable to the type of long-term creep assays that have allowed detection of expansins and yieldins (McQueen-Mason et al., 1992; Okamoto-Nakazato et al., 2000a, b) as, over extended periods, a proportion of the extension does not seem to be reversible (Thompson, 2001).
Furthermore, hysteresis should not lead to artefacts in measurement of stress relaxation of walls, which is also affected by expansin activity (McQueen-Mason and Cosgrove, 1995). While it could be argued that stress relaxation does not involve length change and is therefore not directly related to growth, it is well established that creep and relaxation are closely analogous processes (Aklonis et al., 1972) and there are no clear rheological grounds for rejection of relaxation data.
However (as will be examined in more detail in the next section) it is possible that one-dimensional mechanical measurements generate artefacts and that these are important in the interpretation of both creep and relaxation data.
Non-cellulosic wall components hold microfibrils apart and not together: the plant cell wall is a ‘spaced’ rather than a ‘sticky’ network
In the first papers to show clear images of the architecture of cell wall polymers (by electron microscopy of platinum/carbon replicas produced by rotary shadowing of fast-frozen, deep-etched cell walls) the hemicelluloses between microfibrils were likened to both tethers and ‘pit props’ (McCann et al., 1990) and it was only later that interpretation of these stretches as tethers came to predominate.
The hardening of xyloglucan composites after xyloglucan cleavage (Chanliaud et al., 2004), the aggregation of microfibrils, and the collapse of plant cell wall structure after hemicellulose extraction (McCann et al., 1990) suggest that hemicelluloses bridging the gaps between microfibrils may have a role in maintaining their separation. In addition, the observation that cellulose composite properties were modulated by pectins, but subsequent extraction of the pectins using CDTA did not eliminate the effect (Chanliaud and Gidley, 1999) suggests that the effect of pectins upon composite properties was due to an alteration of the arrangement of the microfibrils, rather than a direct effect of interaction between pectins and cellulose. This behaviour of cellulose composites led Whitney et al. (1999) to suggest that polymer entanglements were involved in retarding extension and that expansins may promote extension by releasing them (Whitney et al., 2000). It has further been suggested that pectins and hemicelluloses increase extensibility by aligning microfibrils so that entanglements are less frequent (Chanliaud and Gidley, 1999; Chanliaud et al., 2004).
However, these effects could also indicate that increased extensibilities of composites were due to the space introduced between microfibrils, rather than an effect of the non-cellulosic polysaccharides themselves. Spacing and alignment effects are not mutually exclusive, but the effects of hemicellulose cleavage or extraction in both composites and plant cell walls suggest that maintaining separation between microfibrils and space within the wall may be important in the behaviour of primary cell walls.
In fact, in models of synthetic polymer behaviour, the free volume between polymer molecules is thought to be fundamental in determining mechanical behaviour. It is well established that polymer mobility can be increased if the volume of free space is increased, simply by the introduction of low molecular weight substances, which are termed plasticizers (Ward and Hadley, 1993) creating substances that are more extensible than the unplasticized polymer. Increased temperature has a similar effect, with clear associations between temperature, mechanical properties, and free volume within the material (Ward and Hadley, 1993). It should be noted that in many plastics (e.g. polypropylene) there is little or no bonding between polymers and in cases where association between polymers increases mechanical strength, it is frequently due to stabilization of ordered fibre structures rather than by formation of a larger scale bonded network (e.g. Kevlar). There is no intrinsic necessity for intramolecular bonding for extended polymeric structures as, in many cases, entanglement or steric hindrance of movement is sufficient.
Such an interpretation of cell wall behaviour is consistent with many properties of plant cell walls, in that cell wall mechanical behaviour would primarily reflect properties of the cellulose microfibrils, but could be modulated by spatial freedom of the microfibrils to move (particularly relative to one another).
In this model, plant cell walls could be visualized as a ‘tangle’ of microfibrils, with wall rheology determined by spatial constraint upon further microfibril movement. Swelling or compaction of the wall could alter the space between microfibrils and, therefore, alter wall properties, perhaps regulated by bridging hemicelluloses, while other non-cellulosic polysaccharides might control microfibril movement by regulating the free volume and the viscosity of the space between microfibrils, acting as controllable plasticizers. In this model, the cell wall could be imagined as being rather like a piece of loosely woven cloth, with the individual threads representing the cellulose microfibrils. When it is stretched the cloth will extend until the threads bunch and retard or halt further extension. If the weave is loose enough, elastic stretching may give way to pulling through of the fibres to bring about a permanent change of shape.
This interpretation is consistent with the behaviour of composites of bacterial cellulose with xyloglucan (Chanliaud et al., 2004) and the aggregation of microfibrils observed after extracting hemicellulose from plant cell walls (McCann et al., 1990). Cell wall loosening as a result of swelling could also be related to observed increases in cell wall thickness associated with wall loosening in fruit ripening (Redgwell et al., 1997) and abscission (Sexton, 1976).
This model has been tested by immersing pieces of frozen and thawed sunflower hypocotyls in mixtures of water with polyethylene glycol (PEG) of high molecular weight (MW). It has been reported that high MW PEG does not penetrate the cell wall (Carpita et al., 1979) and so these solutions are capable of exerting an osmotic pressure upon water in the cell wall and, therefore, modifying the free space (if high MW PEG cannot penetrate the cell wall, the edge of the wall should act as a semi-permeable membrane). As can be seen from Fig. 4, at pH 5.0 an osmotic pressure of 3.0 MPa has a profound effect on creep of plant material. The retardation times were not greatly affected, consistent with the types of processes involved in extension remaining unaltered, but the flow rates of both Kelvin elements were substantially decreased and the viscous flow reduced by an even greater degree (Table 2). Instant rapid extension was observed after returning the material to a pH 5.0 buffer of low osmotic pressure suggesting that penetration of the wall by PEG was limited. Interestingly, the effect was much less marked at pH 6.0, which might suggest some interaction between expansin activity and free space in the wall (results not presented).
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In this type of interpretation, wall properties could be regulated in a number of ways.
- (i) As noted by Veytsmann and Cosgrove (1998), hydrogen bond strength would be expected to affect cell wall volume and not shape. In this light, alterations in hydrogen bonding seem as likely to lead to cell wall swelling or compaction (in turn, affecting cell wall free volume) as length. It is therefore possible that hydrogen bonding between tethers and microfibrils does regulate growth, but by altering free space in the wall and not by directly contributing to mechanical strength. If hydrogen bonding prevents swelling of the wall, expansins might modify the cell wall properties by breaking hydrogen bonds to release steric constraint of microfibril movement and not by breaking load bearing bonds as in the sticky network model. This would accord well with the inhibition of extension and contraction of wall material in non-polar solvents and cell wall swelling observed in fruit ripening and abscission (Sexton, 1976; Redgwell et al., 1997), both of which have been associated with increased expansin activity in other studies (Rose and Bennett, 1999; Cho and Cosgrove, 2000). This was tested using a turbidimetric assay of swelling of cell wall fragments at acidic and neutral pH. Figure 5 illustrates that extinction of suspensions of fragmented cell walls of growing sunflower hypocotyls at a wavelength of 750 nm was 18% higher at pH 4.8 (when expansins would be expected to be active) than in pH 6.2 buffer. Extinction of cell walls suspended in pH 5.0 PEG solution with an osmotic pressure of 1.0 MPa was comparable to that for the pH 6.2 treatment. Extinction is the sum of light absorbance and light scattering, and as all other elements of the suspensions were the same, it seems probable that this effect is due to differences in suspension turbidity, which is related to particle concentration and size. The relative extinctions were constant across the whole range of wavelengths examined (from 450 nm to 750 nm) further suggesting an effect of turbidity rather than absorbance. As the suspensions all derived from the same initial preparation, it seems reasonable to interpret these results as indicating that acidic pH can indeed bring about cell wall swelling.
- (ii) An additional factor in determining the ease of microfibril movement would be the degree to which it is hindered by other molecules in the space between them (effects that would be considered as viscosity at a macro scale). This is likely to be affected by many factors including size, branching, and cross-linking of polysaccharides or other cell wall components. The observation that polysaccharide hydrolases and reduced hemicellulose molecular weights are often associated with increased growth rates (Hoson, 1990
; Loopstra et al., 1998) could be explicable in these terms (although the effect might be balanced by a reduced effect on spacing), as well as the effect of calcium chelators.
- (iii) Free volume within the wall may also be affected by tissue water relations, a possibility raised by Passioura who noted that drought stress might limit the movement of proteins in the cell wall (Passioura, 1994
). The –3.0 MPa water potential used in the PEG treatment of cell wall material described above is of primarily theoretical relevance (although such water potentials might occur in a handful of extreme environments), but some effect on cell wall biomechanical properties has also been observed using PEG solutions with osmotic pressures of 1.0 MPa (DS Thompson, unpublished observations). This type of effect could be ameliorated to some degree by increasing solute concentrations in the cell wall if such solutes were separated from the xylem by locations at which vascular sheaths directed water movement via a symplastic route (as proposed by Canny, 1990).
- (ii) An additional factor in determining the ease of microfibril movement would be the degree to which it is hindered by other molecules in the space between them (effects that would be considered as viscosity at a macro scale). This is likely to be affected by many factors including size, branching, and cross-linking of polysaccharides or other cell wall components. The observation that polysaccharide hydrolases and reduced hemicellulose molecular weights are often associated with increased growth rates (Hoson, 1990
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TopAbstractIntroductionEvidence inconsistent with the...Alternatives to the sticky...ConclusionsReferences |
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In the time since its initial proposal, the interpretation of growing plant cell walls substantially in terms of a load-bearing network of cellulose microfibrils bound together by hemicellulose tethers has come to be the most commonly used description of the growing cell wall (although frequently with some reservations). This paper has suggested that, despite the elegance and explanatory power of this model, it is inconsistent with some lines of evidence and it is also argued that it is hard to imagine how the physical properties of plant cell walls could arise from an isostress network of hemicellulose tethers holding cellulose microfibrils.
It does seem possible that modified versions of the sticky network model would resolve some of these problems, particularly if it proves that hemicelluloses, perhaps particularly xyloglucan, are able to form more robust networks with cellulose than expected. Possible elements of a revised model might be: (i) the binding of some xyloglucan into microfibril stucture (Pauly et al., 1999) and interactions in areas of amorphous cellulose may be stronger than was assumed in calculating the available binding energy. (ii) Microfibrils may be smaller and less regular in shape than was assumed, thus maximizing the surface area available for interaction. (iii) For composites ordered at such small scales, it may not be appropriate to calculate the moduli for isostress systems using moduli for ‘bulk’ materials. One alternative would be that the tethers are extended to the point that they act as ‘taut tethers’ or ‘crystalline bridges’ which would have a higher modulus than amorphous hemicellulose (Peterlin, 1971; Ward and Hadley, 1993). (iv) There may be specific properties of the tether–microfibril interaction that lead to stronger hydrogen bonds being formed than would be usual in an aqueous environment.
A number of alternatives to the sticky network model were also considered. The multi-coat model is similar to the sticky network model, but includes additional interactions between hemicelluloses bound to the microfibril surface and pectins and other hemicelluloses between the microfibrils. Although this did provide explanations for the effect of calcium chelators and reduced relative permittivity, it is not so satisfactory in accounting for work done in extension, unless it is assumed that most of the energy is involved in overcoming interactions with intermediary wall components, rather than bonding at the microfibril surface. In addition, it is also an isostress model and does not provide a good explanation for the high moduli of plant cell walls (at least without incorporating some of the revisions suggested for the sticky network model).
Some workers have also effectively proposed rejection of any genuine relationship between the measured mechanical properties of growing plant cell walls and tissue extension (Nolte and Schopfer, 1997). Because this model denies that most of the data considered in the discussions above are valid, it can resolve the questions raised (essentially by concluding that they were not useful questions in the first place). It remains to be seen whether this alternative interpretation will displace the ‘Lockhartian’ paradigm, but the processes of long-term creep and stress relaxation are very much what might be expected within an interpretation of wall extension as involving ‘plastic’ deformation. It is therefore expected that, although the cleavage of cell wall components by hydroxyl radicals generated by Fenton reactions proposed as an alternative growth mechanism may prove to be important, it is probable that this will be within the current paradigm.
Finally, arguments proposed by a number of previous workers (including Darley et al., 2001; Chanliaud et al., 2004) have been reformulated in terms of theories of synthetic polymer behaviour. In this interpretation, cell wall mechanical properties including cell wall loosening associated with growth are interpreted as arising from the spatial constraint of movement of the cellulose microfibrils, which is regulated by swelling or contraction of the wall to modify the volume of free space between microfibrils or by altering the viscosity of that space. This model seems to account for many aspects of cell wall behaviour and two key predictions, that alteration of wall volume can modify mechanical properties and that cell wall volume will be modified at reduced pH, have been tested and found to be correct. It is suggested that this model may be a useful element in future understanding of how the physical properties of the growing plant cell wall arise from its biochemistry.
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Acknowledgements |
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I owe thanks to Professor Ian Nieduszynski, Professor Bill Davies, Professor Prakash Dey, and Professor Michael Trevan for discussions that have shaped my thinking and to Professor Simon McQueen-Mason for his comments on a draft of the manuscript (although any errors are entirely my own). Bhavita Majevadia and Romeo Radman have helped with some of the experiments. Support for the research has been provided by grants from MAFF, the Nuffield Foundation and the University of Westminster.
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Abbreviations: XET, xyloglucan endotransglycosylase; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid; PEG, polyethylene glycol.
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