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.

The mechanical aspects of cell movement, shape, and growth are of interest across kingdoms ; from the effects of mechanical stresses on cell morphologies and motility in cancer,, to the shaping of plant cells and organs.- Here we focus on the plant kingdom, and the special control of cell shape and growth exerted by the cell wall. The primary plant cell wall is capable of growth, and can be thought of as a mechanical composite, comprised of rigid and strong cellulose microfibrils, embedded in a pectin gel/matrix, and both surrounded and connected by interwoven hemi-cellulose fibers., As new cell wall is produced during growth, an individual wall may also have a multilamellate structure

It is of vital importance that the reader remembers that the cell wall structure just described is idealized and generalized; in truth, we still know very little about the exact structural arrangement of wall components, and we certainly know that cell wall composition varies extensively between cell types, cell ages, tissues, and species,- But if this is the case, how can we ever hope to understand the mechanical behavior of such a structure as it relates to growth? In this addendum to Peaucelle et al., we will present some discussion about the data obtained therein and its biological implications, but also place the methodology in a context with respect to past and future studies of cell wall mechanics.

The Mechanical Properties of Plant Cell Growth and Their Relation to Rheology

Scientists have long been interested in the mechanics of cell growth and its regulation by the cell wall, and subsequently by wall mechanical properties. Mechanically, the cell wall is thought of as viscoelastic. It will behave as an elastic solid, displaying, under a certain stress threshold, instantaneous and reversible deformation (A primer on basic mechanical behaviors can be found in Fig. 1). For example, a cell wall will display elastic deformations due to fluctuations in turgor. Above a certain stress threshold, the cell wall will behave as a viscous fluid. At this point, the cell wall will undergo viscous deformation, leading to deformation of the wall, combined or not with the incorporation of new material into the existing wall structure. The whole process would be viscoplastic, an irreversible deformation with time dependence. For excellent reviews on this topic see refs.,, It is prudent to note that changes in wall chemistry likely effect wall mechanics and growth; the feedback between these processes would result in a very dynamic balance between the cell wall and cell growth. So far this is the theory of mechanical growth, but how are the mechanical properties of cell walls experimentally determined? Rheology refers to the study of the flow of matter, or the behavior of a soft-solid material under applied force; it is concerned with the elastic, viscous, and plastic behavior of materials (Fig. 1). Let us examine how these experimentally derived 'rheological' behaviors, in non-natural conditions, relate to the mechanical properties of the cell wall that regulate growth.

Figure 1. Rheological properties of materials under deformation. (A) Elastic: when an elastic material is deformed by indentation, the indentation and retraction curves are identical. There is no time delay in the material’s response, and the material returns to its undeformed state after retraction (gold arrow). (B) Viscoelastic: when a viscoelastic material is indented, the slope of the retraction curve no longer matches that of the indentation curve. Since the material ‘flowed’ away from the indentation, it requires some time to return back to the undeformed state. The material does eventually return to the undeformed state (gold arrow). (C) Viscoplastic: when a material deforms plastically it does not return to its original state after retraction. This can be seen in the retraction curve, which does not match the indentation start point (gold vs. orange arrow).Indentation curves in Red, retraction curves in Blue. D) Many biological materials display more complex behaviors than those in (A-C) when deformed, either by indentation, stretching, or shearing. A linear elastic material will show a constant relationship between force and deformation (dashed line). Biological materials may exhibit elastic strain stiffening (purple) or viscous shear thickening (green). In these cases the depth or speed of the deformation alter the response, respectively.

The literature is rich with studies that examine the rheological behaviors of plant tissues, organs, and cell wall analogs. The studies using plants have focused on large-scale behaviors as they relate to growth dynamics, but have been limited in their resolution with respect to individual cells within a tissue or the individual components of a single cell wall. In general these studies all involve the application of force to a tissue, by manipulating turgor or by adding physical weight, and monitoring the resulting deformation (Fig. 2, for a methods review see ref.). Examining the relationship between force and deformation over time provides information on elasticity and viscoelasticity. In 1877, Hugo de Vries devised a method to look at turgor induced elastic expansion in cell walls, with which he correlated an increase in bulk elastic wall behavior with increased growth rates in sunflower hypocotyls. The growth promoting hormone auxin (in aerial tissues) induced increases in wall viscoplasticity as shown by Heyn and later decomposed by Cleland into both plastic and elastic components. Cessation of growth in rye coleoptiles was correlated with a decrease in plasticity, although Nolte and Schopfer argue that in fact this plasticity is viscoelasticity. Recently, cell wall mutants have enabled a closer look at the mechanical roles of wall components. As an example, mutants lacking xyloglucan display growth defects such as root stunting and dwarfism, which is reflected in the reduced creep of isolated cell walls; however, paradoxically, mutant walls also exhibited increased elastic and plastic deformation.,

Figure 2. Different scales in mechanical experimentation. (A) A classic extensometer where a piece of tissue (green) is fixed between a rigid arm and a deforming load. The deformation of the tissue sample can be measured over time with constant load, or with changing loads. Deformation of whole tissue ranges from millimeters. Adapted from Cosgrove. (B) A diagrammatic representation of the AFM-based experimental methods of Milani et al. and Peacuelle et al. as applied to shoot apical meristems (i, shaded yellow). The indentations of Milani (ii) were performed with a 40 nm pyramid indenter, to a depth of ~50 nm. This method is thought to provide information on a small section of the cell wall only. In contrast, the methods of Peaucelle (iii) with 1μm or 5 μm diameter spheres and ~500 nm indentation depths, provide information on larger cell wall sections and several tissue layers, respectively. Predicted 'depth of information' in B colored by rainbow gradients.

Mechanical studies of cell wall analogs allow for insights into the contributions of various wall components to mechanical behavior without compensation in composition by the plant. Non-plant cellulose networks, produced by Gluconacetobacter xylinus (Acetobacter xylinus),exhibit extreme strength in tensile tests, and this strength is lessened by the addition of pectins or hemicellulose into the structure. Interestingly, the strength of the composite remained lesser after removal of all pectin by enzymatic digestion. It is possible that this tempering of strength by the pectin matrix and the interwoven hemicellulose is required for a material to be permissive of growth. Indeed, in this analogous material, viscoelastic behavior (as creep) was only seen when hemicellulose was included in the composite. Pectin gels also exhibit interesting material properties on their own, such as strain-stiffening, a characteristic of biological gels. the more you deform them the more force you need to apply to keep the deformation increasing (Fig. 1). A similar phenomenon may occur in viscoelastic materials, shear thickening, where the viscosity increases with increasing deformation (Fig. 1). It has recently been suggested that strain-stiffening of cell walls may contribute to regulation of growth rates in plant tissue.

New Methods and New Scales

So far, the experiments introduced have dealt with composite behaviors of both wall components and multiple cells within tissues. In order to examine the cell wall properties of individual cells and composite tissues newer methods of rheological testing were required. Microindentors have been used to study cell mechanics in pollen tubes and onion epidermal cells but these equipments are limited in their force range and spatial resolution. In a recent study, we have presented an Atomic Force Microscopy (AFM)-based method for measuring cell wall elastic and viscoelastic behavior at cellular and tissue levels. This study was immediately preceded by a complementary AFM-based method developed by Milani et al. Together these two methods provide a unique look at the mechanical properties of cell walls, with the potential for unprecedented mechano- structural resolution.

Here we must introduce a vitally important concept: when examining the relationship between force and deformation, everything is relative! The data obtained will be relative to the time of the experiments, the scale of deformation, and the magnitude of forces applied (Fig. 2). As an illustration, the rapid indentations of 0.2s in Peaucelle et al. provide purely elastic information, whereas indentations held for 10 sec provided information on stress-relaxation i.e., viscoelastic behavior. This can also be seen when contemplating the deformation scales used in Milani et al. (~50 nm), compared with those in Peaucelle et al. (250–500 nm) and the micrometer and millimeter deformations measured in previous methods (Fig. 2)., One of the exciting implications of this physical reality is that by altering the type of indentation, one could gather data from different layers of a composite tissue and also from within a multi-lamellate cell wall itself., For the rest of this addendum, we will focus on the information obtained in Peaucelle et al. its scale and implications for cell wall behavior, and its relevance to the mechanics of growth.

What is Being Measured, and What Does it Mean?

In Peaucelle et al., the elasticity measured for cell walls was strongly influenced by the pectin matrix. Manipulations of the pectin matrix chemistry were shown to alter organ outgrowth patterning at the shoot apical meristem, and we were able to correlate these chemical changes in pectin methylesterification levels with changes in the coefficient of elasticity (herein referred to as EA, the apparent Young’s modulus) of the cell wall. Because of the time delay between the induction of chemical modification and measurement of EA (12+ hours), it is possible that the alteration of pectin structure could have led to mechanical changes in other wall components, which also contributed to decreases in EA; however, pectin modification is either a major contributor to the EA measured, or acts as a trigger for this mechanical change. Future work aims to discover how much of these measurements can be directly attributed to which wall component. For now, we will focus on what we know about pectins and growth.

Mutants in pectin amount or composition display changes in rheology. In addition, changes in pectin chemistry are correlated with growth ability in hypocotyls., So how could changes in pectin structure, and resulting changes in elastic rheological properties, be affecting growth? There are several possible ways for pectin chemistry to affect growth. First, the pectin matrix may mechanically affect the movement of other cell wall polymers, as suggested by Abasolo et al. The authors put forth a model whereby the stiffness/density/elasticity of the pectin matrix alters the ability of hemicellulose chains to unravel under extension forces. This may be extended to a conceivable effect of cellulose microfibril movement within the wall as well. Second, the pectin matrix itself may provide mechanical strength to the cell wall composite. As mentioned earlier Ca2+-cross-linked pectin gels exhibit strain stiffening., Strain stiffening may be a mechanism through which growth is controlled, although at this point it is unclear whether the load born by the pectic matrix would be biologically relevant in this context given the presence of major-load bearing cellulose fibers, which would also exhibit strain stiffening. Third, alterations in pectin chemistry would lead to changes in the porosity of the pectin matrix. This may be exhibited in either the elasticity of the wall, or in its viscoelastic behavior if water movement within the pectin gel contributes to this parameter. Because the pectin gel matrix is not a solid, but a porous material, changes in its porosity would affect water movement and conceivably the movement of wall-modifying agents such as expansin, XTH, or endoglucanases. Fourth, the hydration and swelling of the cell wall matrix, which is sensitive to pectin methylation status, could also affect the mechanical properties of the cell wall, perhaps by altering the interactions of other cell wall polymers. The hydration status of the cell wall may also conceivably affect enzyme activity and movement. And lastly, demethylesterification of pectins releases hydrogen ions into the cell wall, which cause an acidification which in turn may activate other wall modifying agents.

Conclusions

It is apparent from the literature, and from our recent work, that there can be a strong correlation between rheological elasticity and growth; however, it remains to be seen what components of the wall contribute to these changes and how they mechanistically control these behaviors. It is obvious that changes in pectin matrix chemistry have a more profound effect on growth than generally assumed,,, but we should not neglect the known importance of hemicellulose and cellulose as well.

The recent introduction of AFM-based micro and nanoindentation methods, have allowed higher spatial resolution that ever before—we can now examine single cell wall behavior and localized tissue behavior in planta, and are no longer limited to whole organ data. But as introduced here, when examining the relationship between force and deformation: everything is relative! This means that while new methods increase our understanding of the mechanical cell wall and add more questions, they provide a different and complementary set of data to more established methods. We must utilize mechanical data obtained at all length and time scales, if we truly wish to understand the mechanics of the cell wall with respect to growth.