Using SAXS to reveal the degree of bundling in the polysaccharide junction zones of microrheologically distinct pectin gels

The results of microrheological studies carried out on ionotropic pectin gels, particularly the manifest power law behavior observed at high frequencies, indicate that by using different assembly conditions gels can be formed in which the elementary network strands have different stiffnesses. It has been hypothesized that these differences reflect different network architectures, the extreme cases of which might be described as (i) dimeric calcium-chelating junction-zones of limited extent, linked by considerably longer, flexible, single-chain sections, or (ii) semiflexible bundles consisting of extensively aggregated dimeric junction zones that latterly become entangled and cross-linked. To test this hypothesis directly, microrheologically distinct pectin gels have been generated using different assembly modalities, in particular by using different concentrations of polymer and cross-linking ions and by contrasting the controlled-release of ions or ion-binding groups, and the resulting systems have been studied by small-angle X-ray scattering. The results straightforwardly reveal that gels that are clearly more semiflexible from a microrheological point-of-view contain larger scattering entities than those with a more flexible character. Furthermore, a more detailed interpretation of the scattering data with the aid of molecular modeling suggests that for the gels formed here those with a semiflexible microrheological signature consist predominantly of network filaments consisting of four or more chains, whereas those with a more flexible signature are predominantly single-chain sections linked by dimeric associations with no more that a few percent of the chains bundled to any higher extent. The ability to generate differing network architectures from the same polymer that fulfill different functional requirements, either in vivo in the plant cell wall, where pectin plays a crucial structural and mechanical role, or in vitro in a myriad of applications, makes these biomimetic biopolymer networks of considerable interest.     

Microstructure and rheological behavior of pure and mixed pectin gels

The microstructure and the rheological properties of pure HM (high methoxyl) and LM (low methoxyl) pectin gels and of mixed HM/LM pectin gels have been investigated. Gel formation of either the HM or LM pectin, or both, was initiated in the mixed gels by varying the sucrose and Ca(2+) content. The microstructure was characterized by transmission electron microscopy, light microscopy, and confocal laser scanning microscopy. HM and LM pectin gels showed aggregated networks with large pores around 500 nm and network strands of similar character. Small differences could be found, such as a more inhomogeneous LM pectin network with shorter and more branched strands of flexible appearance. LM pectin also formed a weak gel in 60% sucrose in the absence of calcium. A highly inhomogeneous mixed gel structure was formed in the presence of 60% sucrose and Ca(2+) ions, which showed large synergistic effects in rheological properties. Its formation was explained by the behavior of the corresponding pure gels. In the presence of 60% sucrose alone, a homogeneous, fine-stranded mixed network was formed, which showed weak synergistic effects. It is suggested that LM pectin interacts with HM pectin during gel formation, thereby hindering secondary aggregation leading to the aggregated networks observed for the pure gels.     

Mechanical properties of primary plant cell wall analogues

Mechanical effects of turgor pressure on cell walls were simulated by deforming cell wall analogues based on Acetobacter xylinus cellulose under equi-biaxial tension. This experimental set-up, with associated modelling, allowed quantitative information to be obtained on cellulose alone and in composites with pectin and/or xyloglucan. Cellulose was the main load-bearing component, pectin and xyloglucan leading to a decrease in modulus when incorporated. The cellulose-only system could be regarded as an essentially linear elastic material with a modulus ranging from 200 to 500 MPa. Pectin incorporation modified extensibility properties of the system by topology/architecture changes of cellulose fibril assemblies, but the cellulose/pectin composites could still be described as a linear elastic material with a modulus ranging from 120 to 250 MPa. The xyloglucan/cellulose composite could not be modelled as a linear elastic material. Introducing xyloglucan into a cellulose network or a cellulose/pectin composite led to very compliant materials characterised by time-dependent creep behaviour. Modulus values obtained for the composite materials were compared with mechanical data found for plant-derived systems. After comparing bi-axial and uni-axial behaviour of the different composites, structural models were proposed to explain the role of each polysaccharide in determining the mechanical properties of these plant primary cell wall analogues.     

Rheological study into the ageing process of high methoxyl pectin/sucrose aqueous gels

The ageing process of high methoxyl pectin (HMP)/sucrose gels was followed at different ageing temperatures by small amplitude oscillatory experiments. Dynamic mechanical measurements allowed the characterisation of the point at which the system undergoes the sol/gel transition. The HMP/sucrose system is extremely sensitive to temperature variation during ageing, especially in the lower temperature range. The viscoelastic behaviour through the gel point changes with the ageing temperature, probably due to variations in mobility of the pectin chains, and consequently, in the lifetime of junction zones. Weaker pectin networks are formed under thermal conditions unfavourable to the development of hydrophobic interactions. Gel time and elastic modulus have a complex dependence on temperature, which could be attributed to the different thermal behaviour of the intermolecular interactions that stabilise the nonpermanent cross links of these physical networks.     

Material properties of concentrated pectin networks

We have examined the mechanical behaviour of different types of pectin at high concentrations (> 30% w/w), relevant to the behaviour of pectin in the plant cell wall, and as a film-forming agent. Mechanical properties were examined as a function of counterion type (K(+), Ca(2+), Mg(2+)), concentration and extent of hydration. Hydration was controlled in an osmotic stress experiment where pectin films were exposed to concentrated polyethylene glycol [PEG] solutions of known osmotic pressure. We investigated the mechanical behaviour under simple extension. The results show that the swelling and stiffness of the films are strongly dependent on pectin source and ionic environment. At a fixed osmotic stress, both Ca(2+) or Mg(2+) counterions reduce swelling and increase the stiffness of the film.     

In vitro synthesis and properties of pectin/Acetobacter xylinus cellulose composites

Pectin and cellulose are major components of most primary cell walls, yet little is known about the way in which they interact either during assembly or in subsequent functional performance of the wall. As a mimic of cell wall assembly, we studied the formation of molecular composites formed by deposition of cellulose from Acetobacter xylinus into pectin/calcium systems, and the molecular, architectural and mechanical properties of the composites obtained. The formation of interpenetrating cellulose/pectin composite networks (as envisaged in current models for primary cell walls) required a pre-existing, but not too strong, pectin network. For pectin either in solution or strongly networked, phase separation from cellulose occurred, providing two physical models for the formation of middle lamellae. Composite networks showed no evidence of direct molecular interaction between the components, but pectin networks became more aggregated following deposition of cellulose into them. The shear strength under small deformation conditions for cellulose/pectin composites was very similar to that of cellulose alone. In contrast, under uniaxial tension, extensibility was greatly increased and stiffness decreased. These major changes were due to the effect of pectin on cellulose network architecture at deposition, as they were maintained upon removal of the pectin component. These results show that the presence and physical state of pectin at the time of cellulose deposition in muro may be a significant determinant of subsequent extensibility without compromising strength.     

Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. Implications for pectin methyl esterase action, matrix properties, and cell adhesion

Homogalacturonan (HG) is a multifunctional pectic polysaccharide of the primary cell wall matrix of all land plants. HG is thought to be deposited in cell walls in a highly methyl-esterified form but can be subsequently de-esterified by wall-based pectin methyl esterases (PMEs) that have the capacity to remove methyl ester groups from HG. Plant PMEs typically occur in multigene families/isoforms, but the precise details of the functions of PMEs are far from clear. Most are thought to act in a processive or blockwise fashion resulting in domains of contiguous de-esterified galacturonic acid residues. Such de-esterified blocks of HG can be cross-linked by calcium resulting in gel formation and can contribute to intercellular adhesion. We demonstrate that, in addition to blockwise de-esterification, HG with a non-blockwise distribution of methyl esters is also an abundant feature of HG in primary plant cell walls. A partially methyl-esterified epitope of HG that is generated in greatest abundance by non-blockwise de-esterification is spatially regulated within the cell wall matrix and occurs at points of cell separation at intercellular spaces in parenchymatous tissues of pea and other angiosperms. Analysis of the properties of calcium-mediated gels formed from pectins containing HG domains with differing degrees and patterns of methyl-esterification indicated that HG with a non-blockwise pattern of methyl ester group distribution is likely to contribute distinct mechanical and porosity properties to the cell wall matrix. These findings have important implications for our understanding of both the action of pectin methyl esterases on matrix properties and mechanisms of intercellular adhesion and its loss in plants.     

Biological solutions to transport network design

Transport networks are vital components of multicellular organisms, distributing nutrients and removing waste products. Animal and plant transport systems are branching trees whose architecture is linked to universal scaling laws in these organisms. In contrast, many fungi form reticulated mycelia via the branching and fusion of thread-like hyphae that continuously adapt to the environment. Fungal networks have evolved to explore and exploit a patchy environment, rather than ramify through a three-dimensional organism. However, there has been no explicit analysis of the network structures formed, their dynamic behaviour nor how either impact on their ecological function. Using the woodland saprotroph Phanerochaete velutina, we show that fungal networks can display both high transport capacity and robustness to damage. These properties are enhanced as the network grows, while the relative cost of building the network decreases. Thus, mycelia achieve the seemingly competing goals of efficient transport and robustness, with decreasing relative investment, by selective reinforcement and recycling of transport pathways. Fungal networks demonstrate that indeterminate, decentralized systems can yield highly adaptive networks. Understanding how these relatively simple organisms have found effective transport networks through a process of natural selection may inform the design of man-made networks.     

Probing the mechanical contributions of the pectin matrix: Insights for cell growth

The plant cell wall has a somewhat paradoxical mechanical role in the plant: it must be strong enough to resist the high turgor of the cell contents, but at the right moment it must yield to that pressure to allow cell growth. The control of the cell wall's mechanical properties underlies its ability to regulate growth correctly. Recently, we have reported on changes in cell wall elasticity associated with organ formation at the shoot apical meristem in Arabidopsis thaliana. These changes in cell wall elasticity were strongly correlated with changes in pectin matrix chemistry, and we have previously shown that changes in pectin chemistry can dramatically effect organ formation. These findings point to a important role of the cell wall pectin matrix in cell growth control of higher plants. In this addendum we will discuss the biological significance of these new observations, and will place the scientific advances made possible through Atomic Force Microscopy-based nano-indentations in a relatable context with past experiments on cell wall mechanics.     

Diffusing wave spectroscopy microrheology of actin filament networks.

Filamentous actin (F-actin), one of the constituents of the cytoskeleton, is believed to be the most important participant in the motion and mechanical integrity of eukaryotic cells. Traditionally, the viscoelastic moduli of F-actin networks have been measured by imposing a small mechanical strain and quantifying the resulting stress. The magnitude of the viscoelastic moduli, their concentration dependence and strain dependence, as well as the viscoelastic nature (solid-like or liquid-like) of networks of uncross-linked F-actin, have been the subjects of debate. Although this paper helps to resolve the debate and establishes the extent of the linear regime of F-actin networks' rheology, we report novel measurements of the high-frequency behavior of networks of F-actin, using a noninvasive light-scattering based technique, diffusing wave spectroscopy (DWS). Because no external strain is applied, our optical assay generates measurements of the mechanical properties of F-actin networks that avoid many ambiguities inherent in mechanical measurements. We observe that the elastic modulus has a small magnitude, no strain dependence, and a weak concentration dependence. Therefore, F-actin alone is not sufficient to generate the elastic modulus necessary to sustain the structural rigidity of most cells or support new cellular protrusions. Unlike previous studies, our measurements show that the mechanical properties of F-actin are highly dependent on the frequency content of the deformation. We show that the loss modulus unexpectedly dominates the elastic modulus at high frequencies, which are key for fast transitions. Finally, the measured mean square displacement of the optical probes, which is also generated by DWS measurements, offers new insight into the local bending fluctuations of the individual actin filaments and shows how they generate enhanced dissipation at short time scales.     

Gelation of high methoxy pectin in the presence of pectin methylesterases and calcium

High methoxy pectin was submitted to various amounts of a fungal pectin methylesterase (PME) from Aspergillus aculeatus and of a plant PME from orange in the presence of calcium. The systems were characterized by rheological means during the gelation process. By the way of in situ demethoxylation with low amount of orange PME, it was possible to gel pectin from the beginning of the reaction although its high degree of methylation around 70. To understand this unusual properties, the behaviour of the two enzymes was investigated in pectic gels and in solution through the analysis of content and distribution of the remaining methyl esters. In the gel, the degree of methylation decreased slowly with orange PME and rapidly with Aspergillus PME. The degree of methylation and degree of blockiness after treatment with each PME in solution or in gels were slightly different. Possible explanations for this are evolving visco-elastic properties, including gel formation or influence of calcium on the enzyme–substrate complex.     

Effects of Ca, Cu, Al and La on pectin gel strength: implications for plant cell walls

Rheology of Ca-pectate gels is widely studied, but the behaviour of pectate gels formed by Cu, Al and La is largely unknown. It is well known that gel strength increases with increasing Ca concentration, and it is hypothesised that this would also be the case for other cations. Pectins are a critical component of plant cell walls, imparting various physicochemical properties. Furthermore, the mechanism of metal toxicity in plants is hypothesised to be, in the short term, related to metal interactions with cell wall pectin. This study investigated the influence of Ca, Cu, Al and La ion concentrations at pH 4 on the storage modulus as a function of frequency for metal-pectin gels prepared from pectin (1%) with a degree of esterification of 30%. Gels were formed in situ over 6 d in metal chloride solution adjusted daily to pH 4. Cation concentration was varied to develop a relationship between gel strength and cation concentration. At similar levels of cation saturation, gel strength increased in the order of La < Ca ? Al ? Cu. The swelling of the gels also varied between cations with Ca gels being the most swollen.     

Pectin and the role of the physical properties of the cell wall in pollen tube growth of<Emphasis Type="Italic"> Solanum chacoense</Emphasis>

The cell wall is one of the structural key players regulating pollen tube growth, since plant cell expansion depends on an interplay between intracellular driving forces and the controlled yielding of the cell wall. Pectin is the main cell wall component at the growing pollen tube apex. We therefore assessed its role in pollen tube growth and cytomechanics using the enzymes pectinase and pectin methyl esterase (PME). Pectinase activity was able to stimulate pollen germination and tube growth at moderate concentrations whereas higher concentrations caused apical swelling or bursting in Solanum chacoense Bitt. pollen tubes. This is consistent with a modification of the physical properties of the cell wall affecting its extensibility and thus the growth rate, as well as its capacity to withstand turgor. To prove that the enzyme-induced effects were due to the altered cell wall mechanics, we subjected pollen tubes to micro-indentation experiments. We observed that cellular stiffness was reduced and visco-elasticity increased in the presence of pectinase. These are the first mechanical data that confirm the influence of the amount of pectins in the pollen tube cell wall on the physical parameters characterizing overall cellular architecture. Cytomechanical data were also obtained to analyze the role of the degree of pectin methyl-esterification, which is known to exhibit a gradient along the pollen tube axis. This feature has frequently been suggested to result in a gradient of the physical properties characterizing the cell wall and our data provide, for the first time, mechanical support for this concept. The gradient in cell wall composition from apical esterified to distal de-esterified pectins seems to be correlated with an increase in the degree of cell wall rigidity and a decrease of visco-elasticity. Our mechanical approach provides new insights concerning the mechanics of pollen tube growth and the architecture of living plant cells.     

The mechanical properties and molecular dynamics of plant cell wall polysaccharides studied by Fourier-transform infrared spectroscopy

Polarized one- and two-dimensional infrared spectra were obtained from the epidermis of onion (Allium cepa) under hydrated and mechanically stressed conditions. By Fourier-transform infrared microspectroscopy, the orientation of macromolecules in single cell walls was determined. Cellulose and pectin exhibited little orientation in native epidermal cell walls, but when a mechanical stress was placed on the tissue these molecules showed distinct reorientation as the cells were elongated. When the stress was removed the tissue recovered slightly, but a relatively large plastic deformation remained. The plastic deformation was confirmed in microscopic images by retention of some elongation of cells within the tissue and by residual molecular orientation in the infrared spectra of the cell wall. Two-dimensional infrared spectroscopy was used to determine the nature of the interaction between the polysaccharide networks during deformation. The results provide evidence that cellulose and xyloglucan associate while pectin creates an independent network that exhibits different reorientation rates in the wet onion cell walls. The pectin chains respond faster to oscillation than the more rigid cellulose.     

Cell wall components affect mechanical properties: studies with thistle flowers

Pollen presentation of Cirsium horridulum depends partially on the thigmonastic contraction of staminal filaments. Although the elastic cuticle is a major component in filament elasticity, it is not clear how the cell wall copes with the shape change. Based on mechanical studies, FT-IR spectroscopy and biochemical analyses we investigated the relationship between cell wall composition and elastic properties using thistle floral tissues as a model. EDTA-extractable pectin correlated with the increased elasticity of the filament and the basal style, suggesting that pectin plays a major role in the elastic behavior of soft tissues. In contrast, covalently linked pectin contributes to the stiffness of the upper style and corolla. Mechanical tests contrasting the soft basal and rigid apical parts of the style after incubation in solutions designed to alter the pectin network confirmed these results. The rigid corolla contained more cellulose than the softer style and filaments. The cellulose-associated xyloglucan of the style and filament cell walls increase the flexibility of cell walls.     

Aluminium effects on mechanical properties of cell wall analogues

Aluminium (Al) toxicity adversely impacts plant productivity in acid soils by restricting root growth and although several mechanisms are involved the physiological basis of decreased root elongation remains unclear. Understanding the primary mechanisms of Al rhizotoxicity is hindered due to the rapid effects of soluble Al on root growth and the close proximity of many cellular components within the cell wall, plasma membrane, cytosol and nucleus with which Al may react. To overcome some of these difficulties, we report on a novel method for investigating Al interactions with Komagataeibacter xylinus bacterial cellulose (BC)‐pectin composites as cell wall analogues. The growth of K. xylinus in the presence of various plant cell wall polysaccharides, such as pectin, has provided a unique in vitro model system with which to investigate the interactions of Al with plant cell wall polysaccharides. The BC‐pectin composites reacted in a similar way with Al as do plant cell walls, providing insights into the effects of Al on the mechanical properties of the BC‐pectin composites as cell wall analogues. Our findings indicated that there were no significant effects of Al (4–160 μM) on the tensile stress, tensile strain or Young's modulus of the composites. This finding was consistent with cellulose, not pectin, being the major load bearing component in BC‐pectin composites, as is also the case in plant cell walls.     

Global structures of high methoxyl pectin from solution and in gels

Images of high methoxyl orange pectin deposited from solution and high methoxyl sugar acid gels (HMSAG) were obtained by atomic force microscopy (AFM) in the tapping mode. For the first time, images of pectin deposited from water revealed that the transition from pectin networks to individual molecules or aggregates thereof occurred at concentrations between 6.5 and 13.1 microg/mL. At 6.5 microg/mL, shapes included rods, segmented rods, kinked rods, rings, branched molecules, and dense circular areas. At 13.1 microg/mL, all of these shapes were integrated into networks. These same structures were discernible in pectin high methoxyl sugar acid gels. Thus one might consider pectin networks in water at concentrations in excess of 10 microg/mL to be separate fluid precursors of networks in high methoxyl sugar acid gels. Examination of AFM images revealed that gels with "uniform" distribution of strands and pores between strands had higher gel strengths as measured by a penetrometer than gels in which strands were nonuniformly distributed and were separated by large and small spaces.     

果胶甲酯酶抑制蛋白(PMEIs)的功能性研究进展

细胞壁是一种复杂的动态网络结构,在植物生长发育、胁迫应答和免疫抗性过程中起着重要的调控和防御作用。果胶(pectin)是细胞初生壁结构中多糖的主要成分之一;其中,同型半乳糖醛酸聚糖(HG)是果胶多糖组分中含量最丰富的线性聚合物。HG的甲基酯化程度变化会导致其酶解形成凝胶,从而影响果胶结构的稳定性。果胶甲酯酶抑制蛋白(PMEIs)通过翻译后机制调控果胶甲酯酶(PMEs)活性,微调果胶多糖甲酯化修饰平衡后,维持细胞壁的完整性和生物力学特性。研究发现,PMEI-PME互作调控果胶甲酯化修饰的稳态是决定细胞黏附、细胞壁硬度和弹性以及器官形态发生的关键因素,同时也是细胞壁应对逆境、释放抗性信号和免疫防御的分子模式。主要对PMEIs在调节植物器官发育过程和应对不同胁迫因子发挥的抗逆功能及调控机制等最新研究进展作出综述。鉴于PMEIs在木本植物中的体内生理活性和调控机制仍有待探索,可为后续填补该领域的研究空白提供理论依据和策略参考。     

Diversity of a Complex Centromeric Satellite and Molecular Characterization of Dispersed Sequence Families in Sugar Beet (Beta vulgaris)

BACKGROUND AND AIMS: The hypothesis was tested that pectin content and methylation degree participate in regulation of cell wall mechanical properties and in this way may affect tissue growth and freezing resistance over the course of plant cold acclimation and de-acclimation. METHODS: Experiments were carried on the leaves of two double-haploid lines of winter oil-seed rape (Brassica napus subsp. oleifera), differing in winter survival and resistance to blackleg fungus (Leptosphaeria maculans). KEY RESULTS: Plant acclimation in the cold (2 degrees C) brought about retardation of leaf expansion, concomitant with development of freezing resistance. These effects were associated with the increases in leaf tensile stiffness, cell wall and pectin contents, pectin methylesterase (EC 3.1.1.11) activity and the low-methylated pectin content, independently of the genotype studied. However, the cold-induced modifications in the cell wall properties were more pronounced in the leaves of the more pathogen-resistant genotype. De-acclimation promoted leaf expansion and reversed most of the cold-induced effects, with the exception of pectin methylesterase activity. CONCLUSIONS: The results show that the temperature-dependent modifications in pectin content and their methyl esterification degree correlate with changes in tensile strength of a leaf tissue, and in this way affect leaf expansion ability and its resistance to freezing and to fungus pathogens.