Journal of Experimental Botany Advance Access published November 25, 2015

Journal of Experimental Botany

doi:10.1093/jxb/erv511

REVIEW PAPER

Plant cell wall extensibility: connecting plant cell growth

with cell wall structure, mechanics, and the action of wall-
modifying enzymes
Daniel J. Cosgrove*
Department of Biology, 208 Mueller Lab, Pennsylvania State University, University Park, PA 16802, USA

Downloaded from http://jxb.oxfordjournals.org/ at Universidad Autonoma de Chiapas on July 11, 2016

*  To whom correspondence should be addressed. E-mail: dcosgrove@psu.edu

Received 18 September 2015; Revised 6 November 2015; Accepted 9 November 2015

Editor: Nadav Sorek, University of California Berkeley

Abstract
The advent of user-friendly instruments for measuring force/deflection curves of plant surfaces at high spatial resolu-
tion has resulted in a recent outpouring of reports of the ‘Young’s modulus’ of plant cell walls. The stimulus for these
mechanical measurements comes from biomechanical models of morphogenesis of meristems and other tissues, as
well as single cells, in which cell wall stress feeds back to regulate microtubule organization, auxin transport, cellu-
lose deposition, and future growth directionality. In this article I review the differences between elastic modulus and
wall extensibility in the context of cell growth. Some of the inherent complexities, assumptions, and potential pitfalls
in the interpretation of indentation force/deflection curves are discussed. Reported values of elastic moduli from
surface indentation measurements appear to be 10- to >1000-fold smaller than realistic tensile elastic moduli in the
plane of plant cell walls. Potential reasons for this disparity are discussed, but further work is needed to make sense
of the huge range in reported values. The significance of wall stress relaxation for growth is reviewed and connected
to recent advances and remaining enigmas in our concepts of how cellulose, hemicellulose, and pectins are assem-
bled to make an extensible cell wall. A comparison of the loosening action of α-expansin and Cel12A endoglucanase
is used to illustrate two different ways in which cell walls may be made more extensible and the divergent effects on
wall mechanics.

Key words:  Atomic force microscopy, Cel12A endoglucanase, cell growth, cell wall, expansin, extensibility, indentation modulus,
Young’s modulus of elasticity.

Introduction
Interest in cell wall mechanics has received a major boost
from recent elegant models of plant morphogenesis in which
mechanical stresses in growing cell walls guide a dynamic
supracellular system that involves microtubule reorganiza-
tion, oriented cellulose deposition, and auxin transport. The
models simulate a highly choreographed process regulating
the location and directionality of cell growth and cell wall
reinforcement in the shoot apical meristem or other tis-
sues or single cells such as the trichome (Laufs et al., 2009;
Kierzkowski et  al., 2012; Robinson et  al., 2013; Bassel
et  al., 2014; Sampathkumar et  al., 2014b; Boudon et  al.,
2015; Yanagisawa et  al., 2015). These ideas, stimulated by
advances in fluorescent tagging of cell components and con-
focal imaging of living shoot apical meristems (Meyerowitz
et  al., 2005; Bainbridge et  al., 2008; Hamant et  al., 2008),
have revived the concepts of biophysical control of meristem
dynamics pioneered by Green (1999) and extended by sub-
sequent advances in cell biology and genetics (Landrein and

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Page 2 of 14 | Cosgrove
Hamant, 2013; Braidwood et  al., 2014). Additional techni-
cal advances include methods based on micro-indentation
and atomic force microscopy (AFM) to assess wall stiffness
at cellular and subcellular resolution in living meristems as
well as in other tissues and single cells (Milani et  al., 2013;
Bonilla et  al., 2015; Braybrook, 2015; Weber et  al., 2015).
Mechanical measures of cell walls are most informative when
interpreted in terms of the structure of cell walls, which are
comprised of relatively stiff cellulose microfibrils embedded
in a hydrated matrix of pectins, hemicellulose, and structural
protein. Concepts of how these components are linked to one
another have changed considerably since early views of the
primary cell wall as a fiber-reinforced polymer composite,
and concepts of cell wall structure are currently in a state of
flux (Cosgrove, 2014b).
Thus it is perhaps timely to review the notion of cell
wall extensibility and how it relates to cell growth, cell wall
structure, the action of wall-loosening processes, and purely
mechanical measures of wall viscoelasticity such as wall stiff-
ness. I use ‘viscoelasticity’ here rather loosely to encompass
the range of material properties of cell walls that include
elastic, viscous, plastic, and retarded elastic deformations,
but not to include enzyme-dependent, or chemorheological,
flows. Although integration of these inter-related concepts
into a comprehensive theory of plant cell growth is still some
way off, recent movement in this direction is refining our
appreciation of plant growth as a subtle and dynamic pro-
cess. Complementing these developments are genetic studies
of ‘cell wall integrity sensing’ (Wolf et  al., 2014; Hamann,
2015; Hofte, 2015), which have identified signal transduction
elements in this defense response, but leave unanswered ques-
tions such as what ‘wall integrity’ means at the structural level
and what is actually being sensed. This topic is also linked
to a resurgence of interest in pectins and their methylation
state in wall structure and wall sensing (Peaucelle et al., 2008,
2011; Palin and Geitmann, 2012; Wolf and Greiner, 2012;
Braybrook and Peaucelle, 2013).
In this review, I  use the term ‘wall extensibility’ to mean
the ability of the cell wall to increase in surface area irrevers-
ibly during growth. Unlike growing bacterial cells, where an
increase in wall surface is directly coupled to addition of new
wall components, plant cells grow in surface area by a spread-
ing movement of cellulose microfibrils and associated matrix
components in the plane of the wall without necessary addi-
tion of new wall polymers (Cosgrove, 2014a). It involves a
coupled process of wall stress relaxation and water uptake,
described below. In the long term, addition of new polymers
is needed to maintain wall integrity, but wall synthesis and
surface expansion are separable processes, at least in cells with
diffuse growth patterns. In tip-growing cells such as the pol-
len tube (Rojas et al., 2011; Sanati Nezhad et al., 2014), wall
synthesis and surface expansion are more tightly coupled and
thus a modified conceptual framework may be needed, which
is beyond the scope of this review, but many of the concepts
discussed here also apply in modified form to tip growth.
Wall extensibility, as used here, is characteristic of grow-
ing cell walls and differs profoundly from elastic (=revers-
ible) extensibility, which is a property of both growing and
non-growing cells, for example when cell turgor pressure is
modulated or when an external force, such as from an AFM
tip, suddenly impinges on a cell. As reviewed two decades
ago (Cosgrove, 1993), wall extensibility depends on the rate
of wall-loosening processes acting on the cell wall to induce
stress relaxation, which creates the conditions for sustained
cell water uptake and irreversible physical enlargement of the
growing cell. A  central message of this update is that wall
extensibility and wall elasticity are not the same, and in some
instances are not correlated with each other. A second mes-
sage is that methods to measure non-elastic properties such as
extensibility of cell walls at cellular resolution in complex tis-
sues need to be developed to probe deeper into the physical,
cellular, and genetic control of plant morphogenesis.
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What is wall extensibility and how is it
measured?
Early concepts of wall extensibility and its relationship to
cell growth were summarized by Heyn (1940) and updated by
Cleland (1971). These ideas, in which cell growth was thought
to result from yielding of a plastic wall to the forces gener-
ated by cell turgor pressure (P), pre-dated the first detailed
molecular model of a growing cell wall (Keegstra et al., 1973)
and did not offer a specific molecular concept of wall extensi-
bility other than the general notion that the physical viscosity
or plasticity of the wall matrix controlled wall extensibility.
Although ample evidence has accrued in the meantime that
this purely viscoelastic concept of cell wall growth falls short
of reality (Taiz, 1984; Cosgrove, 1993), it seems to be the
assumption of many recent studies that use micro-mechani-
cal mapping of surface stiffness to infer morphogenetic con-
trol mechanisms for meristems and single cells.
A major (but often misunderstood) conceptual advance in
the field was taken by Lockhart (1965) who worked out a con-
sistent biophysical theory connecting cell water uptake and
yielding of the cell wall, eventually leading to recognition of
wall stress relaxation as the seminal biophysical event under-
lying cell growth (Ray et al., 1972; Cosgrove, 1985; Hamant
and Traas, 2010). Wall stress relaxation results from rear-
rangements (yielding) of the load-bearing polymers in the cell
wall, consequently reducing cell turgor pressure and creating
the impetus for simultaneous water uptake into the cell, volu-
metric expansion of the cell wall chamber, and restoration of
wall stress (Fig. 1A). The molecular nature of stress relaxa-
tion is key to understanding how plants regulate their growth
and will be discussed below, but a complete understanding of
the mechanism still eludes us, largely because the stress distri-
bution and load-bearing components of the growing cell wall
are not well understood.
Wall stress relaxation in vivo has been measured by use of
the pressure probe, psychrometer, or pressure chamber to
monitor the decay in water potential or turgor pressure in
growing tissues isolated from an external supply of water, a
condition that blocks the uptake of water needed to restore
turgor and wall stress following wall relaxation (Cosgrove,
1987a, b). Under these conditions, turgor pressure ideally
 Wall extensibility and elastic modulus   |  Page 3 of 14

A (1) (2) (3)

Reduced
Turgor Water influx
wall stress
and
pressure
turgor

cell expands

wall stress wall loosening

stress relaxaon

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restoring turgor and wall stress

B Time course for stress relaxaon C Growth rate v. turgor

Turgor pressure

Growth rate
Rate constant = φ εv

0
Y

Time Turgor pressure

Fig. 1.  Biophysics of plant cell growth. (A) Diagram illustrating the concept of wall stress relaxation and its connection with turgor pressure and induced
water flows. (1) In a well-hydrated non-growing cell, the cell reaches osmotic equilibrium, with wall stresses counter-balancing the outward force of turgor
pressure against the wall. (2) Growing cell walls are loosened, resulting in a reduction in cell wall stress and turgor pressure. This means that elastic
elements in the cell wall shrink as a result of the slippage or bond breakage caused by wall loosening. (3) In response to the reduced cell turgor and water
potential, water flows into the cell, elastically expanding the wall and restoring turgor and wall stress. This process is illustrated as discrete steps, but
relaxation, water influx, volume enlargement, and turgor restoration occur simultaneously. (B) Time course for stress relaxation measured, for example,
with the pressure probe in a growing tissue that is isolated from an external water supply. Turgor pressure decays gradually to a yield threshold Y with a
rate constant given by the cell wall extensibility coefficient ϕ times the cell volumetric elastic modulus εv. Based on Cosgrove (1987a). (C) Graph of growth
rate as a function of turgor, idealized in the equation growth rate=ϕ (P–Y), where ϕ is wall extensibility, P is turgor pressure. and Y is yield threshold. From
such a plot, estimates of Y and ϕ may be made.

decays to a steady value, the yield threshold, with a rate
constant that depends on the wall extensibility coefficient
(Fig.  1B). Hence the general notion of wall extensibility is
defined in this formulation with two parameters, the yield
threshold and the extensibility coefficient. These parameters
are also revealed in plots of growth rate as a function of turgor
pressure (Fig. 1C). Actual plots of growth rate versus turgor
are not so linear and display a more gradual transition at the
yield threshold, as does creep of isolated cell walls subjected
to a series of tensile stresses (Takahashi et  al., 2006). Thus
the idealized equation representing the turgor dependence of
the rate of cell wall enlargement [GR=ϕ(P–Y); see Fig.  1C
legend] is only a rough approximation of the actual behavior
of growing tissues. Turgor dependence of plant growth is an
inevitable physical consequence of the facts that (i) wall stress
is essential for stress relaxation (and ensuing water uptake)
and (ii) the stress relaxation rate is a function of the wall stress
itself. When growth is measured as % h−1, extensibility (ϕ) has
units of % h−1 MPa−1, while the yield threshold (Y) is usu-
ally measured in units of cell turgor pressure (MPa). Living
cells may complicate this relatively simple ideal behavior by
modulating wall-loosening processes dynamically in response
to reduced turgor, reduced growth rate, or other conditions
(Green et al., 1977; Nakahori et al., 1991).
Unfortunately, these methods for measuring wall relaxation
do not currently lend themselves to micromechanical map-
ping of meristems. Kierzkowski et al. (2012) and Nakayama
et al. (2012) took a step in this direction by measuring osmotic
Page 4 of 14 | Cosgrove
inflation and deflation of cells on the surface of the shoot api-
cal meristem, but did not map extensibility or yield thresh-
olds. Their approach might be extended by use of a series
of osmotic solutions to calculate growth rate versus turgor
for each surface cell (as in Fig. 1C) and plotting extensibil-
ity coefficient and yield threshold values across the meristem
surface.
Contrary to early ideas that wall growth and extensibility
arise from passive polymer motions of a viscoelastic wall,
various observations indicate that there is more to it and that
it depends on continual wall loosening.
(i)  W hen isolated cell walls are heat treated so as to inacti-
vate wall-bound enzymes and are clamped at constant
force (i.e. in wall creep assays), they undergo only a tran-
sient viscoelastic extension that decays with a half-time
of a few minutes or less, in contrast to the walls of living
cells that extend steadily for many hours or days (Taiz,
1984). The viscoelastic extension is a combination of
elastic and retarded elastic deformations that are distinct
from wall loosening related to growth (Cosgrove, 1993;
Nolte and Schopfer, 1997; Schopfer, 2006). Likewise, the
stress relaxation behavior of inactivated cell walls differs
from that of living cells; compare Cosgrove (1987b) with
Yamamoto et al. (1970).
(ii)  When isolated walls that are not inactivated are clamped
at constant force at acidic pH, they extend steadily
for many hours under the continued action of endog-
enous expansins bound to the cell wall (Cleland et  al.,
1987; Cosgrove, 1989; McQueen-Mason et  al., 1992).
Extensions of 50–100% occur before the walls thin to the
point of breakage. Such breakage does not occur in walls
of living cells, presumably because the wall is continually
reinforced by newly incorporated polymers.
(iii) When respiration of living cells is inhibited by cold or
metabolic inhibitors, growth ceases almost immediately
but the viscoelastic properties of the isolated cell wall do
not change [i.e. as measured by stress/strain assays (Taiz,
1984)]. In a similar vein, when growth is rapidly inhibited
by light (Cosgrove, 1988b) or rapidly stimulated by auxin
(Cleland, 1984), wall viscoelastic properties change only
slowly after the change in growth.
Thus, while growing cell walls clearly have viscoelastic prop-
erties (Taiz, 1984; Schopfer, 2006) and viscoelasticity some-
times tracks changes in wall extensibility (with some delay),
they are distinctive properties. Ray (1987) proposed exten-
sibility to be the result of wall loosening in series with sub-
sequent viscoelastic extension, each governed by separate
rate constants k1 and k2, respectively (Fig. 2). The observa-
tions cited above suggest that k2 is larger than k1. In such a
case, the wall deformation following sudden changes in wall
stress will be (transiently) dominated by purely viscoelastic
extensions (k2) whereas under steady-state conditions wall
enlargement is controlled by loosening (k1). This formula-
tion may oversimplify the behavior of growing cell walls,
but it provides a handy conceptual framework for thinking
of loosening and viscoelasticity as distinctive aspects of the
growing cell wall.
How does wall stiffness relate to wall
extensibility?
With the advent of user-friendly atomic force microscopes
and other micro-indentation devices coupled to microscopes,
reports of cell wall stiffness or modulus for shoot apical mer-
istems, leaf epidermal layers, single cells, and other plant
tissues have become common (Milani et  al., 2013; Routier-
Kierzkowska and Smith, 2013). The principle of the inden-
tation method is deceptively simple: a cantilever or other
mechanical device is maneuvered to the surface of a cell or
tissue, and a force–deflection curve is measured as the tip of
the device makes contact with the surface, pushes against it,
and then retracts. Probes vary from relatively sharp, pyrami-
dal shapes of AFM tips as small as ~4 nm diameter (Xi
et al., 2015) to spherical beads or cylinders with contacts of
1–10  μm diameter (Braybrook, 2015). The deformation is
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typically fast, in the millisecond range for some AFM-based
measurements to seconds for other types of indentation pro-
tocols, so wall loosening during the measurement is unlikely
to affect the force–deflection response. There is a trade-off in
speed: too fast and hydraulic flows of the bathing fluid and
within the wall may influence the deflection rate; too slow and
water outflows from the compressed cells can influence the
process, if the wall is pressed into the cell as in ball tonometry
(Cappella and Dietler, 1999; Lintilhac et al., 2000; Forouzesh
et al., 2013; Beauzamy et al., 2015).
While the appearance of such force–deflection curves may
resemble the curves generated by uniaxial stress–strain assays
of hypocotyl or coleoptile wall specimens (i.e. in an ‘Instron’
tester) (Cleland, 1967), such resemblance belies the many com-
plications inherent in micro-scale indentation of living plant
surfaces. One major difference is the direction of the applied
force: a tensile force in the plane of the cell wall, as used in
a uniaxial tensile tester, is resisted by cellulose microfibrils in
the direction of the force, whereas this is not the case for an
indentation force normal to the plane of the wall for indenta-
tion assays. Some of the assumptions and complicating issues
of the indentation measurements have been discussed in the
literature (McKee et al., 2011; Milani et al., 2013; Braybrook,
2015). Tip size, shape, and penetration depth can influence
whether the probe penetrates into the cell wall, deforms the
cell wall locally, or changes the whole cell shape (and adjacent
cells) more globally. Usually it is unclear how the deflection
is partitioned into these three different aspects of wall dis-
placement. Extracting information about cell wall material
properties (usually quantified as a modulus or its reciprocal, a
compliance) from indentation measurements always involves
fitting the force–deflection data to some kind of model and
making assumptions about wall structure, cell shape, and the
influence of turgor pressure, which stretches the cell wall and
applies a pre-stress to the load-bearing wall polymers.
Turgor pressure is an important factor to be considered
because it increases the effective stiffness of the cell, much
as air pressure in a bicycle tire stiffens the tire (stiffness
measures the resistance to deformation). There are at least
three potential components to this effect: (i) non-linearity in
the stress–strain curve (‘strain hardening’), which generally

wall loosening
sff wall extensible wall
k k
1 2
(stress relaxaon)
Fig. 2.  Conceptual model of Ray (1987) linking cell wall loosening and wall viscoelasticity with rate constants k1 and k2 for biochemical and viscoelastic
steps. This concept was formulated for wall extension but can be extended for parallel hydraulic steps (shown in the bottom line), with stress relaxation
originating from wall loosening and subsequent water uptake driving the passive viscoelastic extension.
increases with wall strain; (ii) resistance to indentation due
to pre-stresses in the plane of the wall (‘tightening of the
drum head’); and (iii) resistance stemming from the outward
force of turgor pressure, when the wall is perceptibly pressed
into the cell (the latter is the basis for tonometry assessments
of turgor). Non-linear stress–strain behavior has long been
known in the field of plant wall relations, where the cell volu-
metric elastic modulus generally increases as turgor pressure
increases (see, for eample, Buchner et  al., 1981; Tomos and
Leigh, 1999). Separating turgor effects from inherent wall
properties can be done, but may be complicated, involving
osmotic treatments, finite element modeling of the cell, and
many assumptions for tractable mechanical analysis (Hayot
et  al., 2012; Forouzesh et  al., 2013; Bonilla et  al., 2015;
Digiuni et al., 2015; Weber et al., 2015). For surface indenta-
tion measurements, the tensioning effect of turgor is likely
to have a large effect on the force–deflection curves, even at
small deflections. It is for this reason that some AFM-based
studies have plasmolyzed the shoot apical meristem before
measurement of wall mechanics (e.g. Peaucelle et al., 2011).
While this procedure removes complications due to turgor, it
also alters the pattern of wall stresses across the meristem and
the mechanical state of the cell walls.
It is also worth noting at this point that there are many
different kinds of moduli in the world of plant cell walls, and
they measure different properties. Accessible introductions to
this topic include Wainwright et al. (1976), Niklas (1992), and
Burgert (2006). Generally speaking, a modulus is a measure
of the stress/strain ratio where stress is conventionally defined
as force per unit area and strain as relative change in length,
width, thickness, volume, etc. There are many different ways
to apply a stress and to measure the concomitant strain, and
consequently there are many different kinds of moduli; they
may all be expressed in the same units, but they measure dif-
ferent things and ought not to be mixed up. In the case of the
cell volumetric elastic modulus mentioned above, the modu-
lus (εv) is defined as dP/dV where dP is a change in cell turgor
pressure (MPa) and dV is the relative change in cell volume
(dimensionless). This modulus measures the elastic stretch-
ability of the cell wall chamber and is not the bulk modu-
lus (=1/compressibility) used in mechanics, with which it has
sometimes been confused (Cosgrove, 1988a). When cells lose
water, cell volume and turgor pressure decrease and εv quanti-
fies this relationship. In cells with very stiff walls (high modu-
lus), turgor pressure falls steeply as cells lose water. Values
of εv for non-lignified plant cells typically range from 1 MPa
to 50  MPa (Zimmermann and Steudle, 1978; Cosgrove,
Wall extensibility and elastic modulus   |  Page 5 of 14
viscoelasc
extension
extended, sff wall
(water uptake)
1988a; Tomos and Leigh, 1999). With an εv of 10 MPa, a
cell would lose 1% of its volume for a 0.1 MPa reduction in
turgor pressure. In contrast, the bulk modulus of water (the
main component of plant cells) is ~2000 MPa, so an increase
in atmospheric pressure by 0.1 MPa would compress the cell
negligibly (0.005%).
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A more commonly cited modulus is the Young’s modulus
of elasticity (E), an engineering unit that quantifies the stress/
strain ratio in a uniaxially extended strip of ‘Hookean’ mate-
rial, defined as material displaying linear elasticity (i.e. strain
is proportional to stress and is fully and immediately revers-
ible upon reduction of stress). Growing plant cell walls—with
their hierarchical, multilamellate, and anisotropic arrange-
ment of stiff cellulose microfibrils bonded to each other and
to hydrated matrix polymers—exhibit elastic deformation
plus complicated, non-linear, and time-dependent strains
that fall outside the Hookean ideal. The non-linear character
of plant cell walls during mechanical deformation was high-
lighted in a recent study (Digiuni et al., 2015), but it has been
recognized for many years.
Engineers generally avoid non-linear behavior by working
at small strains (say, <0.1%) where non-linearities become
inconsequential. This is in contrast to the larger strains often
imposed during micro-indentation studies of cell walls and
whole-tissue stress–strain assays. Non-linearity becomes a
troublesome issue when modulus values are calculated from
different parts of a stress–strain curve. The anisotropic con-
struction of growing cell walls necessitates recognition of at
least three distinct elastic moduli (Fig. 3): EL for changes in
length, EW for changes in width, and ET for changes in thick-
ness of a cell wall strip [this is an incomplete description of
the full mechanics of the wall, which is beyond the scope of
this review; see Niklas (1992) and Weber et al. (2015) for more
detailed treatment]. Cellulose microfibrils, which are depos-
ited in the plane of the cell wall, greatly influence EL and EW,
whereas pectins probably exert dominant control of ET (Ha
et al., 1997). The complex structure of plants cell walls means
that these moduli are first-order approximations of the actual
elastic behavior of cell walls. Additionally, the propensity of
growing cell walls to deform irreversibly in a time-dependent
manner under the tensile stresses generated by cell turgor
or external forces complicates their mechanical properties
even further. It is, of course, exactly this non-linear, time-
dependent, irreversible behavior of growing cell walls that is
of greatest relevance for growth and morphogenesis. At low
strains (<0.1% deformation) and rapid measurements, elastic
behavior probably dominates, but at larger strains (>0.1%)
Page 6 of 14 | Cosgrove
EL
ET
EW
Fig. 3.  Schematic diagram of the anisotropic construction of plant cell
walls, made of multiple lamellae (only two are shown), each with aligned
cellulose (longitudinal in the top lamella, transverse the bottom lamella),
and embedded in a hydrated matrix of pectins and hemicellulose. The
three major elastic moduli (EL, EW, ET) govern the stress–strain responses
of walls to forces applied in the three orthogonal axes of the wall. Note that
cellulose microfibrils contribute to EL and EW, but have less influence on ET.
Wall indentation measurements may be most sensitive to ET.
and longer time scales the non-elastic behaviors may become
significant. For an example of the issues involved, see Hansen
et al. (2011) who have reviewed the background for the time
dependence of viscoelastic measurements of cell walls and
described a procedure of dynamic testing for living, but not
growing, potato explants.
Notwithstanding the foregoing complications, Young’s
elastic moduli of cell walls based on indentation assays are
commonly reported. From the force–deflection curves gener-
ated by micro-scale indentations of plant cell surfaces, one
may estimate an elastic modulus by fitting the data to an
appropriate model. The type of model depends on the size of
the tip, the depth of probe penetration into the wall, and the
kind of wall deformation that is envisaged.
For nanometer-scale measurements made with a typi-
cal atomic force microscope using a sharp probe (~4–40 nm
diameter), a common assumption is that wall deformation
is local, that large-scale bending of the cell wall is minimal,
and that the influence of turgor pressure on cell wall stiff-
ness is negligible. The force–deflection curve is then fitted to
a mechanical model, usually a variant of the Hertz–Sneddon
model (Sneddon, 1965) or the DMT model (Derjaguin et al.,
1975), to estimate an elastic modulus of the deformed mate-
rial (Routier-Kierzkowska and Smith, 2013; Braybrook,
2015). Complications in the analysis and interpretation of
the complex curves obtained with living cells are outlined by
Bonilla et al. (2015) who describe an empirical approach to
distinguish different modes of wall deformation that occur
during whole-cell indentation with small tips.
In a pioneering report of micro-mechanical mapping
of the shoot apical meristem, Milani et  al. (2011) observed
that reduction of cell turgor pressure by addition of 0.25 M
NaCl had no effect on mechanical measurements, at least
within the error of the measurement. This seems a perplex-
ing result because tensioning of the wall by turgor pressure
should make the wall appear stiffer. The authors estimated
the wall deformation of their measurements to be ~300 nm
in diameter. This is ~20-fold larger than the spaces between
cellulose microfibrils, which are believed to bear much of
the turgor-generated wall stress; thus I  would expect a pro-
nounced turgor dependence of the indentation curves. The
lack of turgor dependence in their measurements suggests to
me that the AFM tip did not probe the mechanically relevant
(load-bearing) portion of the cell wall. Because the outer part
of the cell wall is the oldest region, it has undergone very large
extensions in the course of its lifetime, possibly fragmenting
its structure and uncoupling it from turgor-generated tensile
stresses in the plane of the wall. This ought to be the case
for the outer epidermal wall in the shoot apical meristem,
but probably not for internal cell walls within the meristem
that are relatively young (i.e. formed during a relatively recent
cell division). This interpretation would be consistent with
the concept summarized by Taiz (1984) that the inner half
of the cell wall bears turgor-generated wall stresses and that
the outer half may not be important for wall enlargement.
If true, it casts doubt on the interpretation of AFM meas-
urements that mechanically probe only the outermost part of
the epidermal wall. Differences in the young and old regions
of the wall could lead to differential contraction and shear
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stresses across the wall when turgor pressure is reduced, but
apparently this possibility has not been studied in a quantita-
tive manner.
Turgor dependence of plant cell stiffness is reflected in the
everyday experience that carrots become limp upon dehy-
dration and has been documented many times: 50 years ago
as measurements of whole-tissue modulus (Virgin, 1955;
Nilsson et al., 1958), a generation later as measurements of
single-cell εv (Huesken et al., 1978; Buchner et al., 1981), and
more recently in micro-indentation studies (Wang et al., 2004;
Hayot et al., 2012; Forouzesh et al., 2013; Weber et al., 2015).
In the later set of studies, large-scale wall deformations were
employed and the deflection data were fit to mechanical mod-
els of whole-cell deformation. A detailed example illustrating
the modeling that is required and the assumptions involved
in such models is the analysis of tobacco cell suspension
cells indented with a cylindrical probe of ~3 μm diameter by
Weber et al. (2015). Two elastic moduli for these cylindrical
cells were estimated: EL (153 MPa) for changes in cell length
and EW (571 MPa) for changes in cell circumference (the third
modulus, for cell wall thickness, was not estimated). Using
these elastic moduli and assuming the cells are ideal cylin-
ders with a turgor pressure of 0.6 MPa, I  estimated εv for
these cells to be ~15 MPa, which is within the range of values
reported in the literature. Change in length made the largest
contribution to the change in volume for the εv estimate. This
calculation serves as a check that the wall moduli reported
in this study are consistent with the expected pressure–vol-
ume relations of such cells and also gives a point of compari-
son for values of EL, EW, and εv. Wang et al. (2004) provide
another point of comparison for spherical tomato suspen-
sion cells compressed between two surfaces. They report a
Young’s modulus of 2.3 GPa and an estimated εv of 7 MPa.
They considered the wall to be isotropic and 126 nm thick
and they also took into account the outflow of water from the
cell when it is compressed in this way (during the 1 s compres-
sion). A theoretical sensitivity analysis performed by Weber
et al. (2015) showed that their calculations were particularly
sensitive to estimates of cell radius, indentation depth, and
cell wall thickness, assumed to be 1250 nm, which is ~10-fold
thicker than estimates from other studies of suspension cells
(Samuels et al., 1995; Sabba et al., 1999; Wang et al., 2004).

Re-parameterization of geometrical quantities reduced some
of these dependencies. One has to wonder whether the ~10-
fold difference in Young’s moduli in these two studies is a
reflection of different assumptions of the thickness of the
cell walls rather than differences in the material properties of
the walls.
Compared with these estimates of elastic moduli in the
plane of the plant cell wall, elastic moduli based on indenta-
tion using a small probe (10 nm to 1  μm) yield values that
are much smaller, by factors of 10- to >1000-fold (Milani
et al., 2011; Peaucelle et al., 2011; Radotic et al., 2012; Ma
et  al., 2013; Zdunek and Kurenda, 2013; Sampathkumar
et al., 2014a; Wang et al., 2014; Braybrook, 2015). The rea-
son for the wide divergence in indentation values does not
seem to have been explored, but it appears too large to be
based on real differences in cell wall structure. The systematic
difference between in-plane tensile moduli and values from
indentation assays arises in part from the nature of the nano-
indentation method, which applies a force that is normal to
the plane of the cell wall. In other words, they are measur-
ing different properties of an anisotropic wall (note that cell
walls, which are traditionally called isotropic because of a
random direction of cellulose in the plane of the wall, are still
highly anisotropic when the third dimension, wall thickness,
is considered). The calculation of a modulus from force–
deflection curves by micro-indentation typically assumes that
the indented material (i) is isotropic; (ii) shows linear elastic-
ity; and (iii) is >10 times thicker than the depth of indenta-
tion. The construction of epidermal walls deviates from these
assumptions, and mechanical contributions due to cell turgor
pressure add to the difficulty. As noted by Milani et al. (2013),
‘it is unclear which properties of the cell wall are measured,
because the force is roughly perpendicular to cellulose micro-
fibrils’. In cross-sections of dry woody cell walls, where the
nano-indentation method was first applied to plant cell walls,
experimental complications and problems of interpretation
were previously identified (Gindl and Schöberl, 2004). The
mechanical situation for indentations of growing tissues pre-
sents a different set of problems, but I  hazard a guess that
the method at its best measures a type of wall compressive
modulus, dominated by pectin properties, but with some
(poorly defined) influence of the microfibrils at right angles
to the principle force vector. The term Young’s modulus or
even ‘apparent’ Young’s modulus for these measurements
seems misleading. The term ‘indentation modulus’ (Eder
et al., 2013) may be a clearer way of describing these values
and avoiding confusion with Young’s moduli in the plane of
the wall.
From the foregoing discussion, it is evident that the mod-
ulus calculated from micro-indentation measurements dif-
fers from the in-plane tensile moduli of the cell wall. This
disparity may not affect some conclusions based on relative
changes in stiffness across a surface, but it could have sig-
nificant ramifications for dynamical models of plant mor-
phogenesis that depend on use of correct values for in-plane
stresses and tensile moduli of the wall. Chickarmane et  al.
(2010) and Sampathkumar et  al. (2014a) review some of
these models. Additionally, recent studies indicate that the
Wall extensibility and elastic modulus   |  Page 7 of 14
pectin-rich middle lamella can make a substantial contribu-
tion to the mechanical behavior of the multicellular epidermal
surface (Zamil et al., 2014, 2015; Kim et al., 2015). Hence, the
mechanical behavior of a subcellular patch of epidermal cell
wall may not fully account for the mechanics of a multicel-
lular sheet of epidermal cell wall. A related point was made
by Chan et al. (2011) and Crowell et al. (2011) in studies of
cellulose reinforcement of epidermal cell walls in Arabidopsis
hypocotyls. They inferred from microscopic observations (but
not mechanical measurements) that the outer epidermal wall
is not the major determinant of the directionality of hypoco-
tyl growth, which instead was linked to the anisotropic orien-
tation of cellulose in the side walls and inside walls. Previous
work supporting this concept was summarized by Baskin
(2005). Hence, the contribution of internal cell walls to mor-
phogenesis of other growing organs and meristems is worth
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further scrutiny (Baskin and Jensen, 2013), perhaps using
the combination of mechanical measurements and modeling
employed for shoot apical meristems.
Extensibility, cell wall structure, and wall
loosening
A grand challenge in this field is to relate cell wall structure
to the mechanics of cell walls and the action of cell wall-loos-
ening agents that induce wall stress relaxation and provoke
water uptake needed for cell growth. For more than two dec-
ades the dominant concept of the growing cell wall has been
of a scaffold of well-separated cellulose microfibrils mechani-
cally tethered together by xyloglucans and embedded in a
pliant pectin gel (Hayashi, 1989; Pauly et al., 1999; Scheller
and Ulvskov, 2010). Evidence for and against the tethered-
network concept was summarized by Scheller and Ulvskov
(2010), but recent years have witnessed mounting dissatis-
faction with the concept (Cosgrove, 2014b). It falls short in
view of the mild phenotype of xyloglucan-deficient mutants
(Cavalier et  al., 2008; Park and Cosgrove, 2012a), nuclear
magetic resonance (NMR) analysis of matrix–cellulose
interactions in the wall (Dick-Perez et al., 2011; Wang et al.,
2015), and enzymatic probes of cell wall mechanics (Park and
Cosgrove, 2012b). In addition, recent AFM imaging of never-
dried primary cell walls shows that cellulose microfibrils are
not evenly separated, as commonly depicted, but frequently
make close contact with each other, forming junctions or
bundles that may be important for wall mechanics (Zhang
et al., 2014). Cellulose bundles are also seen in cellulose mats
made by Acetobacter xylinus where they may contribute to the
mechanics of these cellulosic hydrogels (Whitney et al., 1999;
Martinez-Sanz et  al., 2015). Thompson (2005) challenged
the tethered-network concept by a calculation that hydrogen
bonding between xyloglucans and cellulose was too weak
to resist turgor-generated wall stresses (however, I  believe
the calculation made questionable assumptions); instead, he
offered a purely polymer-based view of wall extensibility as
controlled by hydration, spacing, and entanglement of matrix
polymers. Although cell wall hydration contributes greatly to
primary wall mechanics (Edelmann, 1995; Tang et al., 1999;
Page 8 of 14 | Cosgrove

Evered et al., 2007; Kennedy et al., 2007; White et al., 2014;
Kim et  al., 2015), the concept seems unlikely to provide a
mechanism by which a cell regulates its growth.
A recent alternative to the tethered-network concept pro-
poses that wall mechanics and wall extensibility are con-
trolled at limited sites where cellulose microfibrils come into
close contact (Park and Cosgrove, 2012b). In agreement with
experimental results, most of the xyloglucan is bound to cel-
lulose but does not serve a load-bearing role under static
conditions. This concept accounts for lack of wall loosening
by xyloglucan-specific endoglucanase and cellulose-specific
endoglucanase, alone or in combination, in contrast to the
effectiveness of bifunctional endoglucanases that cut both of
these wall components (Park and Cosgrove, 2012b). It also
accounts for the lack of appreciable loosening by xyloglu-
can endotransglucosylase (Saladie et  al., 2006). The revised
The biomechanical hotspot model was tested by atomis-
tic simulation (Zhao et  al., 2014), which indicated that the
strength of cellulose–xyloglucan–cellulose junctions may
contribute substantially to the mechanical strength of the cell
wall. This was a nanometer-scale test, but the more interest-
ing mechanical properties of the hotspot model may emerge
from simulation of larger scale behavior of the wall, much
as the mechanical properties of a fishnet depend not just on
the strength of the individual junctions between two ropes
but on the spacing between junctions as well as the thickness
and flexibility of the ropes. A nominal attempt in this direc-
tion was a finite element model (FEM) that simulated a single
lamella of nearly parallel cellulose microfibrils connected by
limited xyloglucan linkers (Nili et al., 2015). The linkers were
implemented in the model as short tethers between micro-
fibrils rather than as the monolayer of xyloglucan adhesive

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model proposes limited, inaccessible junctions (dubbed bio-
mechanical hotspots) where cellulose and xyloglucan chains
are tightly adherent to each other (Fig. 4). Such sites may also
be the target of expansin (Wang et al., 2013). Suggestions of
direct cellulose–cellulose junctions in primary walls may also
be found in earlier reports (Boyd and Foster, 1975; Scheller
and Ulvskov, 2010).
suggested by the enzyme results of Park and Cosgrove (2012b)
and modeled by Zhao et  al. (2014). The study concluded
that relatively few xyloglucan linkers per cellulose microfi-
bril were needed to maintain mechanical stability, but that
additional load-bearing elements were needed to maintain
realistic elastic moduli. This elastic weakness might be elimi-
nated by including multiple lamellae with cellulose oriented

Fig. 4.  An artistic rendition of the arrangement of cellulose, xyloglucan and pectins based upon recent results (Cosgrove, 2014b). Cellulose microfibrils
(~3 nm diameter) are shown in blue and their arrangement is traced from an AFM image of the most recently deposited surface of an onion epidermal
cell wall (Zhang et al., 2014). Xyloglucans (green) are shown as random coil structures interspersed with pectins and with limited contacts with cellulose
(based on NMR results). Pectins (yellow) are shown as two forms, a rigid form in contact with cellulose microfibrils and a more mobile form filling the
spaces between microfibrils. Potential cellulose–cellulose junctions are highlighted in red. Image adapted from Current Opinion in Plant Biology, 22C,
Cosgrove DJ. Re-constructing our models of cellulose and primary cell wall assembly, 122–131, Copyright (2014), with permission from Elsevier.

in different directions in the lamellae. The authors speculated
that pectins might contribute to mechanical strength of the
wall, but pectins were not included in their simulation and
likewise were missing in another FEM of a multilamellate cell
wall (Kha et al., 2010). One problem is that the mechanism by
which pectins may interact with cellulose is still unclear, an
issue that merits deeper study, as discussed next.
Pectins
The recent re-evaluation of the role of xyloglucans in cell
wall models coincides with renewed attention to the role of
pectins (Palin and Geitmann, 2012; Peaucelle et  al., 2012;
Wang et  al., 2012; Wolf and Greiner, 2012; Braybrook and
Peaucelle, 2013). There are diverse views on the structure
and the mechanical role of pectins in growing cell walls. The
dominant and traditional view imagines that they form a
soft, gel-like, viscous matrix in which the cellulose–hemicel-
lulose network is embedded, but otherwise not binding cel-
lulose (Carpita and Gibeaut, 1993; Cosgrove, 2005). In this
view, pectins may contribute to cell wall stiffness during
rapid deformations, but their viscosity would cause pectin-
borne stresses to decay rapidly, displacing forces onto more
rigid components of the cell wall. If the gel were to become
sufficiently rigid, for example by calcium-cross-linking of
unesterified regions of homogalacturonan, it could in theory
lock the wall into an inextensible state; however, it is unclear
whether this actually occurs. Pectin de-esterification is likely
to affect many physico-chemical properties of the wall.
A different view, first proposed in the pioneering molecular
model of the cell wall by the Albersheim group (Keegstra
et al., 1973; Albersheim, 1975), considers pectins to be part
of a massive macromolecular matrix that contains covalently
linked domains of pectin, hemicellulose, and protein and that
extensively binds to cellulose surfaces through xyloglucan
domains. Although this model was largely abandoned in the
1980s in favor of the tethered network concept (Fry, 1989;
Hayashi, 1989; McCann et al., 1990), linkages between pec-
tin and xyloglucan or pectin and proteins have been reported
recently (e.g. Popper and Fry, 2008; Tan et al., 2013). A recent
review (Park and Cosgrove, 2015) concludes that the abun-
dance of pectin–xyloglucan linkages is low in most plant tis-
sues, with the notable exception of cell suspension cultures.
Low abundance, however, does not necessarily mean they are
insignificant for wall mechanics. This point remains an open
question.
Unlike xyloglucans, which bind tightly and irreversibly to
cellulose (Hayashi, 1989), pectins bind to cellulose relatively
weakly or not at all, at least during binding assays in vitro
(Zykwinska et  al., 2005, 2008). Lin et  al. (2015b) reported
weak, reversible pectin binding to bacterial cellulose pellicles,
also called hydrogels because they are made of a loose, ran-
dom entanglement of cellulose fibrils containing ~99% water.
The pectin interactions with cellulose were weak and included
components of mere inclusion and entrapment within the
large free space of the entangled cellulosic mat. Neutral pec-
tins (arabinan, galactan) showed stronger interactions than
homogalacturonan (Lin et al., 2015a), consistent with the in
Wall extensibility and elastic modulus   |  Page 9 of 14
vitro binding results cited above. Pectin contributed to the
compression resistance of the cellulose–pectin composite by
limiting hydraulic flows out of the composites, whereas xylo-
glucan interactions with the cellulosic pellicles were more sta-
ble and altered the pellicle compressibility in different ways
(Lopez-Sanchez et  al., 2015). Mobility-resolved 13C-NMR
indicated that the pectin molecules were highly mobile, not
tightly bound to cellulose.
These results with artificial composites are consistent with
the common view that pectins are held in the wall only weakly,
but are in contrast to 13C-NMR studies of native pectin in
plant primary cell walls, which show abundant pectin–cellu-
lose cross-peaks in 2D cross-polarization measurements, indi-
cating extensive, stable pectin–cellulose interactions (Wang
et al., 2012, 2015). As judged by solid-state NMR, cellulose
contacts with pectins were more abundant than contacts with
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xyloglucan. Moreover, although pectins are the most mobile
polymers in the primary cell wall, nearly half of the pectin
was in a rigid form, with a 13C T1 relaxation time similar to
that of cellulose. These results, supported by previous obser-
vations (e.g. Ryden and Selvendran, 1990; Foster et al., 1996),
were interpreted to mean that a substantial pectin compo-
nent in native cell walls is stably bound to cellulose surfaces
or entrapped within cellulose bundles. However, the molecu-
lar nature of the cellulose–pectin interaction still is not clear
and needs further investigation. Moreover, these results do
not establish whether or not pectins have a significant static
load-bearing role in cell wall mechanics.
In biomechanical assays (creep assays), wild-type
Arabidopsis cell walls showed a relatively modest loosen-
ing effect when treated with pectin-degrading enzymes, with
a somewhat larger effect in a xyloglucan-deficient mutant
(Park and Cosgrove, 2012a). The results indicated that pec-
tins do not bear static loads in wild-type cell walls, but may
take on a larger mechanical role in the absence of xyloglucan.
One limitation of this enzymatic approach is that if pectins
are entrapped among microfibril bundles, they might not be
accessible to enzymatic digestion, potentially missing a cryp-
tic load-bearing component. Moreover, the essential role of
pectins in cell–cell adhesion (i.e. the middle lamella) limits
the extent of pectin weakening treatments feasible for these
creep experiments where the sample is held under constant
tensile load (multicellular wall samples fall apart when pec-
tins are significantly degraded). This technical limitation may
be circumvented by micro-indentation assays with isolated
cell walls. In such assays with onion epidermal walls, Xi et al.
(2015) used a sharp pyramidal probe and a Hertz model to
calculate an elastic modulus from the force–deflection curves.
Removal of calcium with a chelator reduced wall stiffness,
decreasing the calculated indentation modulus from 23 MPa
to 16 MPa, presumably as a result of reduced ionic cross-link-
ing of homogalacturonan within the wall (the middle lamella
was not probed). This result confirms that pectins contribute
to the compression resistance of cell walls to fast deforma-
tions. This still leaves open the question of their contribution
to static tensile loads and to slow creep in the plane of grow-
ing cell walls. For comparison, the tensile (Young’s) modulus
of comparable wall material was estimated as 400 MPa (Kim
Page 10 of 14 | Cosgrove
et al., 2015)—a clear example of the difference between the
indentation modulus and tensile modulus of the wall.
In an elegant study of fast and reversible polymer motions
in onion epidermal walls stretched in the plane of the wall,
Wilson et al. (2000) combined dynamic mechanical analysis
with polarized infrared (IR) spectroscopy. They concluded
that homogalacturonan motions were uncoupled in time
from cellulose and xyloglucan motions. To explain this tem-
poral shift, they suggested that homogalacturonan motions
were dominated by strains in the middle lamella which were
subsequently transmitted to cellulose in the cell wall proper.
A subcellular version of this experiment that eliminated the
pectin signal from the middle lamella would be informative
for the question of pectins as load-bearing elements of the
wall proper, particularly if time constants for decays in wall
stress could be assessed for the different structural compo-
nents of the primary cell wall. From dynamic mechanical
measurements of (living) potato explants in combination with
a mathematic model, Ulvskov et al. (2005) inferred that pec-
tins can transmit dynamic, but not static, stresses to cellulose.
Turning to purely computational approaches, Dyson et al.
(2012) developed a noteworthy mathematical model of cell
wall yielding based on continuum mechanics. Their model
considers hemicellulose and pectin to be acting as co-load-
bearing elements, modeled with a mathematical function giv-
ing strongly non-linear stress/strain rate behavior resembling
a yield threshold. The yield threshold was simulated by cross-
links that stretch up to a limit, then break suddenly, with a
role for pectins in the post-breakage yielding. The model
mimics a number of the behaviors of growing cell walls; by
its nature it cannot offer insights into the structural or bio-
chemical bases for wall stress relaxation, but it does present
an attractive approach for further efforts to relate structural
models of cell walls to their mechanical behaviors beyond
simple elastic moduli.
Wall loosening
As a final point, it is instructive to compare the wall-loos-
ening actions of α-expansin and Cel12A, a family-12 endo-
glucanase that cuts both cellulose and xyloglucan (Table 1).
These are the only two classes of proteins known to induce
stress relaxation and creep of plant cell walls, although
it should be noted that Cel12A is a fungal enzyme and we
Table 1.  Comparison of the characteristics of cell wall loosening
by α-expansins and Cel12A, based on their activity with cucumber
hypocotyl walls, based on published results (Yuan et al., 2001;
Park and Cosgrove, 2012b)
Property α-Expansin Cel12A
Hydrolytic action No Yes
Increased plasticity No Yes
Increase elasticity No Yes
Induces creep Yes Yes
Lag time for induction of creep Seconds 6 min to >60 min
do not know of plant enzymes with similar wall-loosening
action. Characterizations of two family-9 endoglucanases
from plants indicate that they have a wide substrate specificity
(Ohmiya et al., 1995; Yoshida and Komae, 2006), suggesting
a wall-loosening action similar to Cel12A, but such action
has not actually been documented. In support of this notion,
ectopic expression of one of these enzymes in Arabidopsis
promoted plant growth (Park et al., 2003), and transcript pro-
filing identified several family-9 genes that are expressed in
growing tissues of grasses (Buchanan et al., 2012).
Cel12A loosens plant cell walls by its hydrolytic activity,
whereas no enzymatic activity has been found for α-expansin.
Both proteins induce creep of isolated, heat-inactivated cell
walls from cucumber hypocotyls and other plant materials,
but with very different kinetics: α-expansin induces creep
within seconds after application whereas Cel12A induced
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creep only after a substantial lag time that depended on
enzyme concentration: a minimum of 6 min at saturating
enzyme concentration, >60 min for low enzyme concentra-
tions (Yuan et al., 2001). Cel12A reduced the elastic and plas-
tic moduli of cucumber walls in uniaxial tensile tests, whereas
α-expansin did not change these moduli. Note that the plas-
tic modulus in these assays measured the irreversible compo-
nent of uniaxial extension, which Nolte and Schopfer (1997)
have re-interpreted to be the result of delayed viscoelasticity
rather than true plasticity. Under this interpretation, Cel12A
increases the viscoelastic hysteresis in the stress–strain curves,
whereas α-expansin does not. By either interpretation, the
results suggest that α-expansin can cause stress relaxation
and creep by a mechanism (sliding at cellulose junctions?)
that does not perceptibly alter cell wall structure, whereas
the cutting action of Cel12A substantially reduces the num-
ber or strength of cellulose–cellulose contacts, resulting in a
mechanically weaker cell wall. Attempts to simulate cell wall
creep by cutting cross-links (e.g. Dyson et al., 2012) may not
properly mimic expansin’s loosening activity, so a refined
model may be needed.
The loosening action of α-expansin bears some resem-
blance to the ‘stick–slip’ or ‘molecular Velcro’ model of
plastic deformation of wet wood (Keckes et  al., 2003;
Cosgrove and Jarvis, 2012; Adler and Buehler, 2013) in
which hydrogen-bonded contact regions between cellu-
lose layers slip and then stick again, restoring mechanical
strength until local stresses exceed the yield threshold and
another local slip event occurs. In woody tissues this is a
purely physical process, whereas in growing cells the selec-
tive slippage, relaxation, and creep of primary walls are
mediated by expansins (and potentially other agents). The
fact that α-expansin can loosen cell walls without reducing
the plastic or elastic moduli is a likely explanation for the
fact that wall extensibility and wall viscoelasticity are often
uncorrelated, as discussed above. Another example of such
a discrepancy is a micro-indentation study of plasticity
along the growing root of Arabidopsis thaliana (Fernandes
et  al., 2012). Plasticity was measured as hysteresis in the
approach and retraction phases of the force–deflection
curves. This is a micro-scale version of the hysteresis seen in
uniaxial tests of cell walls, which is the basis for estimating

elastic and plastic moduli (Cleland, 1967, 1971). In the
study by Fernandes et al. (2012), no correlation was found
between the ‘index of plasticity’ on the living root surface
and the growth rate along the root axis, despite the fact
that cell growth and wall extensibility vary greatly along the
axis (Pritchard, 1994). It might be useful to try this micro
method with wall samples known to vary in wall plastic-
ity (or delayed viscoelastic hysteresis), for example after
Cel12A treatment, to validate that the method can indeed
detect useful plasticity values for cell walls.
Concluding remarks
There is a growing recognition that stresses and strains in the
growing cell wall feed back to cellular systems to regulate
the cytoskeleton, vesicular trafficking of auxin transporters,
and the cellular machinery involved in cellulose deposition.
This provides the impetus for insightful dynamical models
connecting wall mechanics with microtubule organization
and studies to identify the molecular connections of these
elements of the model. Careful thought needs to be given
to the meaning of wall moduli based on surface indentation
assays. They differ from the in-plane tensile elastic modu-
lus, which is the parameter of greatest value for assessing
the stress/strain properties of the cell wall. A deeper under-
standing of wall deformations—both reversible and irre-
versible—will require more extensive experimental testing in
combination with quantitative models of how the structural
components of the cell wall are linked to one another and
what kinds of polymer motions occur during rapid, revers-
ible deformations versus the slow irreversible enlargement
of the growing wall.
Acknowledgements
Preparation of this review was supported by the US Department of
Energy, Office of Science, Basic Energy Sciences as part of The Center for
LignoCellulose Structure and Formation, an Energy Frontier Research
Center under Award # DE-SC0001090.
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