Download Cellulose orientation determines mechanical anisotropy in onion

Journal of Experimental Botany, Vol. 57, No. 10, pp. 2183–2192, 2006
doi:10.1093/jxb/erj177 Advance Access publication 23 May, 2006
RESEARCH PAPER
Cellulose orientation determines mechanical anisotropy in
onion epidermis cell walls
D. Suslov and J-P. Verbelen*
Department of Biology, University of Antwerp, Campus Drie Eiken, Universiteitsplein 1, 2610 Wilrijk, Belgium
Received 21 November 2005; Accepted 1 March 2006
Abstract
The role of cellulose microfibril orientation in determining cell wall mechanical anisotropy and in the control of
the wall plastic versus elastic properties was studied in
the adaxial epidermis of onion bulb scales using the
constant-load (creep) test. The mean or net cellulose
orientation in the outer periclinal wall of the epidermis
was parallel to the long axis of the cells. In vitro cell wall
extensibility was 30–90% higher in the direction perpendicular to the net microfibril orientation than parallel
to it. This was the case for the size of the initial deformation occurring just after the load application and
for the rate of time-dependent creep. Loading/unloading
experiments confirmed the presence of a real irreversible component in cell wall extension. The plastic component of the time-dependent deformation was higher
perpendicular to the net cellulose orientation than parallel to it. An acid buffer (pH 4.5) increased the creep
rate by 25–30% but this response was not related to
cellulose orientation. The present data provide direct
evidence that the net orientation of cellulose microfibrils confers mechanical anisotropy to the walls of
seed plants, a characteristic that may be relevant to
understanding anisotropic cell growth.
Key words: Allium, cell wall extensibility, cellulose orientation,
confocal microscopy, creep test.
Introduction
Plant cells are surrounded by the stiff yet extensible cell
walls that play a crucial role in determining the rate and
direction of cell expansion and in the control of plant
shape in general (Hernandez and Green, 1993; Cosgrove,
2000a). The link between the wall properties relevant for
growth and the rate of steady-state plant cell expansion was
established by Lockhart (1965) and expressed in the
equation that holds true if water transport to the growing
cell is not limiting:
r = ð1=VÞðdV=dtÞ = UðP YÞ
where r is the relative growth rate measured as a change in
cell volume (1/V)(dV/dt); U is the wall yielding coefficient;
P is the turgor pressure; and Y is the yield threshold, the
minimum turgor required for growth. According to Lockhart,
cell expansion occurs because the effective turgor pressure
(P–Y) irreversibly stretches the cell wall.
The parameters U and Y depend on physical (mechanical) properties of the cell wall and on the metabolic
reactions modifying the interactions between the wall
constituents (Taiz, 1984; Cosgrove, 1993). The mechanics
of a cell wall are determined by its structure that resembles
that of composite materials (Kerstens et al., 2001). The
walls consist of stiff semi-crystalline cellulose microfibrils
cross-linked by hemicelluloses and embedded in a gel-like
matrix of pectins (Cosgrove, 1997).
Cellulose microfibrils have traditionally been considered
as the main load-bearing components in the cell wall whose
orientation determines the direction of preferential wall
extension (Carpita and Gibeaut, 1993). This statement was
mostly based on the observation that, in growing cells,
cellulose microfibrils are usually transversely oriented to
the vector of the cell extension (Baskin et al., 1999;
Sugimoto et al., 2000). At the same time, there have been
surprisingly few direct confirmations that a specific alignment of cellulose microfibrils confers to the cell walls a
mechanical anisotropy which is then translated into a
preferential direction of cell growth. Such evidence was reported in early work on cell walls of Nitella, a green alga
(reviewed in Taiz, 1984). It is, however, not obvious
that these data can be extrapolated to higher plants. Nitella
walls are many times thicker than primary walls of higher
plants and, accordingly, contain many more microfibril
* To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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2184 Suslov and Verbelen
layers across the wall thickness (Baskin et al., 1999). Also
this alga, to our knowledge, is not considered as ancestral to
higher plants, and there is no reliable information on its wall
architecture. Searching for appropriate seed plant models
to study the relationship between microfibril orientation
and mechanical cell wall anisotropy has led to the use of
onion and Kalanchoë epidermal peels (Kerstens et al.,
2001). With an Instron-like assay, stress–strain curves for
these tissues were determined. It was demonstrated for the
first time for higher plants that a net parallel orientation of
cellulose microfibrils in the onion epidermis wall coincided with a high mechanical anisotropy. In contrast, the
Kalanchoë epidermis had a random net orientation of
microfibrils and was mechanically isotropic.
The present study confirms and extends the data obtained
in the previous work (Kerstens et al., 2001) using the creep
(constant-load) method for measuring cell wall extensibility
in vitro. Unlike the stress–strain assay, the creep method
mimics the action of turgor on growing cell walls (Taiz,
1984). Due to this fact, data on creep analysis have been
related to the parameters U and Y from the Lockhart equation
(Okamoto and Okamoto, 1994; Büntemeyer et al., 1998;
Thompson, 2001). A similar attempt in relation to the net
microfibril orientation was undertaken in this work. Another
advantage of the constant-load test is its ability to distinguish
between irreversible (plastic) and reversible (elastic) components of cell wall deformation. About 70 years ago, it was
postulated that the plastic component of cell wall extensibility in vitro correlates with the rate of plant cell expansion
in vivo (Heyn, 1932). This point of view was questioned
by Schopfer and co-workers, who claimed that the in vitro
plastic extensibility of isolated cell walls in fact represents
retarded elastic wall extension (Hohl and Schopfer, 1992;
Nolte and Schopfer, 1997). The present study confirms the
presence of a real irreversible component in the extension
of onion epidermal cell walls. Using the advantages of the
creep test, it was also possible to investigate for the first
time the effect of net microfibril orientation on plastic
versus elastic cell wall properties in higher plants.
Materials and methods
Plant material
Onion (Allium cepa L. cv. Bonkajuin) bulbs were commercially
acquired. The bulbs came from an early harvest at the beginning of
June and had not reached their maximal mature size. In the field,
they had some potential for further growth at the moment of harvesting. Strips of adaxial epidermis, 25–30 mm long and 4 mm
wide, were peeled from the equatorial part of the adaxial side of
the third (from the outside) live scale in directions parallel and perpendicular to the axis of the bulb. These ‘longitudinal’ and ‘transverse’ epidermal strips were immediately frozen in liquid nitrogen,
stored at 20 8C, and used within 1 week after isolation.
Analysis of the net cellulose microfibril orientation
The net or mean orientation of cellulose microfibrils in the outer
periclinal wall of the adaxial epidermis was determined using polar-
ization confocal microscopy and fluorescent Congo Red staining as
described previously (Verbelen and Stickens, 1995; Verbelen and
Kerstens, 2000). Thawed epidermal strips were stained in a 1% (w/v)
solution of Congo Red (Sigma) in water for 2 h. After rinsing in
water, the Congo Red-mediated fluorescence of cell walls was
studied using a confocal laser scanning microscope (Bio-Rad MRC
600 mounted on a Zeiss Axioskop) with a 320 (NA 0.5) dry objective and a co-axial rotating object table.
The dichroic fluorescent dye Congo Red preferentially absorbs
light directed parallel to the dipole moment of its chromophoric
group. It has an ability to bind specifically to b-1,4-linked glucan
polymers, especially to cellulose fibrils, and the dipole moments of
Congo Red chromophoric groups will be aligned in the same
direction as the cellulose polymers. If the cellulose fibrils have some
preferential orientation, Congo Red fluorescence will be maximal
when the vector of the polarized exciting laser beam is parallel to
the net cellulose orientation. When the vector is perpendicular
to the net microfibril orientation, the fluorescence intensity will be
minimal. In the case of a net random cellulose orientation, the
wall fluorescence intensity will be equal for all orientations of the
polarized light vector.
The extent to which cellulose microfibrils were aligned with
respect to the cell’s long axis was expressed as the axiality ratio. This
is the ratio between the wall fluorescence intensities with the vector of
the excitation light parallel and perpendicular, respectively, to the cell
longitudinal axis. The axiality ratio equals unity if the net cellulose
microfibril orientation is random. It will have values above or below
unity in walls with, respectively, a net cellulose orientation parallel
or perpendicular to the cell axis. The fluorescence intensity of
Congo Red-stained cell walls was quantified using Photoshop 4.0
software (Adobe).
Extensiometry
In vitro extensibility of onion epidermal cell walls was studied with
a custom-built constant-load extensiometer similar to that described
by Kutschera and Schopfer (1986). Wall extension was measured
using a displacement transducer (model 200HR, Schaevitz, Hampton,
VA, USA) coupled to an in-line amplifier (model MC-IP-15B-B10
from ALTHEN GmbH, Kelkheim, Germany) and a data acquisition
system (model DI-700 from DATAQ, Akron, OH, USA) connected
to a PC. The cell wall strain curves were recorded and analysed with,
respectively, WinDaq/Lite and WinDaq Waveform Browser software
(both from DATAQ, Akron, OH, USA).
The constant loads used in the creep test should ideally induce the
same stress in the isolated walls as that existing in vivo. It is very
difficult, however, to measure the real force extending the epidermal
cell wall in an onion bulb because of its complex nature. This force
is summed from the turgor of epidermal cells, the tension between
the epidermis and the underlying parenchymatic cells (Kutschera
et al., 1987), and the pressure developing between the adjacent scales
of the bulb as a result of their growth. Therefore, the strategy in
this work was to use a maximally broad range of loads to model the
effect of any possible magnitude of the unknown cumulative force
on the extension of the epidermis.
The lever of the extensiometer with the upper clamp attached to its
left shoulder was pre-equilibrated before securing a cell wall sample.
Thereafter, the lever was held in the horizontal position by a lock
under its right shoulder while an initial load was placed on the scale
of the extensiometer. The lower clamp consisted of two vertical
plexiglass plates with two U-shaped rubber rims stuck on their internal surfaces opposite to each other. Closing the clamp gripped the
lower end of the cell wall sample and formed a water-tight chamber around it. This chamber was filled with 10 mM sodium citrate
buffer (pH 6.0, if not otherwise stated) so that the cell wall sample
was completely immersed in the buffer during the measurement.
Cellulose orientation and wall mechanics
2185
A 10 mm segment of an epidermal strip was secured between the
clamps. Cell wall extension was induced by unlocking the lever. A
rapid, practically instantaneous cell wall extension referred to as
‘initial deformation’ was measured 1 s after the load application. It
was followed by a slow, time-dependent deformation (creep) whose
kinetics are represented as an average rate for the intervals 1 s–5 min,
5–10 min, and 10–15 min after the load application (Fig. 2).
To study the reversibility of cell wall extension in vitro, 2 or 5 g
loads were subsequently added onto and then removed from the
scale of the extensiometer at 60 s intervals. On the basis of the
recorded cell wall strain curves, total deformation, initial deformation, and creep were calculated for every step of loading and
unloading. Total deformation (DT) is the change in wall length
between subsequent increases or decreases in load. Initial deformation (DI) is the change in wall length measured 1 s after
loading (unloading). Creep (C) is the amplitude of the timedependent cell wall deformation from 1 s to 60 s after the load
application (removal). Thus, DT=DI+C. To estimate total deformation reversibility, the sum of total deformations at all steps of
unloading-induced cell wall contraction was divided by the sum
of total deformations at all steps of loading-induced cell wall
extension. The result was expressed as percentage reversibility
(elasticity). A similar procedure was used to calculate separately
the reversibilities of creeps and initial deformations. The sum of
creeps (initial deformations) at all steps of unloading was divided
by the sum of creeps (initial deformations) at all loading steps
and expressed as a percentage of creep (initial deformation)
reversibility.
Results
Quantification of the cellulose microfibril orientation in
the outer epidermal cell wall
The onion bulbs used in this study contained two or three
dead dry scales and from four to seven live scales that
surrounded several juvenile ‘bulblets’. The long axes of
epidermal cells in all scales were parallel to the axis of the
bulb. A confocal image of a typical Congo Red-stained
cell from the adaxial epidermis of the third (from outside)
live scale is illustrated in Fig. 1. Fluorescence intensity is
coded in shades of grey. It is clear that when the vector of
the laser beam is parallel to the cell’s long axis (Fig. 1A),
fluorescence in the cell wall is much stronger than when
the vector is perpendicular to the cell axis (Fig. 1B). The
cellulose microfibrils thus have a net longitudinal orientation in this tissue. Analysis of 100 epidermal cells selected
at random from 10 different onion bulbs has established
that the net cellulose alignment in the outer epidermal wall
of the third scale was characterized by the axiality ratio
of 1.5260.04 (SE).
All scales located inward from the third scale also had
a clear longitudinal net orientation of cellulose microfibrils
in the outer periclinal walls of their adaxial epidermis. The
two outermost live scales had only a slightly longitudinal
or even a random net orientation of microfibrils in the
corresponding wall. The third scale was chosen for further
analysis because it was the largest scale with a net longitudinal cellulose orientation. In the terminology used subsequently, ‘longitudinal strips’ refer to epidermis samples
Fig. 1. Mean cellulose microfibril orientation in the adaxial epidermis of
the onion bulb scale. Polarization confocal micrographs of epidermal cell
walls stained with Congo Red. The vector of polarization of the laser is
indicated by arrows. The intensity of fluorescence is coded in shades of
grey from low (black) to high (white). Its values for the outer periclinal
wall of the cell in the centre of the figure are shown as numbers in the
lower left corners of the micrographs. The fluorescence intensity is
maximal (A) and minimal (B) with the vector of polarization parallel and
perpendicular, respectively, to the cell long axis. This shows that the net
orientation of cellulose microfibrils is parallel to the longitudinal axis of
the cell. Scale bar=50 lm.
where the load is applied parallel to the net cellulose
orientation and thus also parallel to the axis of the cell. In
‘transverse strips’, the load is applied perpendicular to the
net cellulose orientation.
2186 Suslov and Verbelen
The in vitro cell wall extensibility depends on the net
cellulose microfibril orientation
Applying a constant load to an epidermal strip resulted in an
immediate high-amplitude initial deformation that was
followed by a slow, time-dependent creep (Fig. 2). It is
clear that extension is much larger transverse to the
cellulose orientation than parallel to it. The rate of creep
always decreases with time during 60 min of measurement
(data not shown). Analysis of the deformation curves
revealed that the whole range of the loads used (5–20 g)
induced stronger extension in the transverse than in the
longitudinal strips (Fig. 3). This difference was highly
significant (P <0.01) for the initial deformations (Fig. 3A)
and the creep rates during the intervals 5–10 and 10–15
min after load application (Fig. 3C, D), where a practically
2-fold difference was registered for the 5 g and 10 g loads.
Thus the isolated epidermal cell walls are much more
extensible in the direction perpendicular to the mean orientation of cellulose microfibrils than in the direction parallel
to the net cellulose orientation.
Interestingly, the difference between the initial creep
rates (0–5 min) of longitudinal and transverse strips was
smaller (compare Fig. 3B with C and D). At 20 g load, it
was even statistically insignificant. The reason for this
discrepancy became evident through analysis of creep
during the first seconds after the load application (Fig. 4).
The dynamics of creep of longitudinal and transverse strips
were absolutely identical during the first 10 s after the
20 g load application. This was due to the non-linear behaviour of the stress–strain dependence in transverse strips
(Fig. 4). For the later time intervals, the dependence of
creep rates on the applied forces was linear in longitudinal
as well as in transverse epidermal strips (Fig. 3B–D).
A 25 g load caused rupture in ;50% of the transverse
strips but never in longitudinal strips. This fact confirms the
Fig. 2. Dynamics of extension of an isolated onion epidermis with
longitudinal (thick line) and transverse (thin line) net orientation of
cellulose microfibrils. Typical curves recorded after a 15 g load application (arrow) are represented. Vertical dashed lines indicate the time
intervals (1 s–5 min, 5–10 min and 10–15 min after the load application) during which the average creep rate was determined.
Fig. 3. Dependence of extension on the load in onion epidermal walls
with longitudinal (thick lines, closed circles) and transverse (thin lines,
open circles) net orientation of cellulose microfibrils. Comparison of
initial deformations (A) and creep rates during the intervals 1 s–5 min
(B), 5–10 min (C), and 10–15 min (D) after the load application. These
values were calculated after analysis of experimental curves of the type
indicated in Fig. 2. Data are means 6SE (n=13).
Cellulose orientation and wall mechanics
Fig. 4. Initial creep rates (1–21 s after load application) of onion
epidermal walls with net longitudinal (thick lines) and transverse (thin
lines) orientation of cellulose microfibrils. Each curve is the average
of 10–12 experiments with different epidermal strips at a given load.
For convenience of comparison, the starting points of the curves are
superimposed at time zero. Notice that for 20 s of measurement, 5 g
increments in the load induced similar increments in extension in
longitudinal strips but decreasing increments in transverse strips.
This indicates that the dependence of the initial creep rate on the load
is non-linear in the transverse orientation of cellulose microfibrils.
Error bars are the SE.
higher tensile strength of the epidermal tissue in the direction
parallel to the net orientation of cellulose microfibrils.
Plastic and elastic components of in vitro extensibility
depend on the net microfibril orientation
Experiments were set up to find out if a true plastic
component of in vitro extensibility exists in onion epidermal walls and, if so, to determine the effect of cellulose
microfibril orientation on plastic versus elastic behaviour
of isolated cell walls.
In a first approach, cell wall deformation was recorded
during a single 1 min loading step followed by complete
unloading. Irrespective of the load used, the epidermal cell
walls immediately returned to their original length after
unloading (not shown). This phenomenon was, however,
not due to the elastic behaviour of the cell wall. It just
reflected the fact that the lever of the extensiometer (see
Materials and methods) was pre-equilibrated before the
measurement and fixed horizontally. After the complete
unloading, the lever came back to its original horizontal
equilibrium position, returning the secured cell wall sample
to the point it occupied before stretching. A single preloading step allowed this artefact to be separated from the
real elastic cell wall properties. In this approach, extension
experiments start from epidermis samples that are kept
under slight tension. As an example, Fig. 5 depicts an
experiment performed with 2 g loads. Epidermal strips
were pre-loaded with 2 g for 1 min. Then an additional
2 g load was applied for 1 min. After its removal, the extension was measured during the next 20 min (Fig. 5). For
2187
Fig. 5. Plastic component in the extension of epidermal walls with net
longitudinal (thick lines) and transverse (thin lines) orientations of
cellulose microfibrils. Epidermal strips were pre-loaded with a 2 g load
(+L) for 1 min. Thereafter an additional 2 g was applied (+L, loading)
and removed 1 min later (–L, unloading). Changes in the subsequent
tissue deformation were recorded for 20 min longer. After unloading,
a significant part of tissue deformation remains, demonstrating the presence of a real plastic component (Ep) in onion epidermal wall deformation. Typical curves are shown.
both orientations of the cellulose fibrils, the unloading resulted in an instant elastic shrinkage of the epidermal strips,
followed by a limited (not more than 5 lm) retarded elastic
contraction, that lasted 2–3 min. Thereafter, the length of
the epidermal strips did not change any more (Fig. 5). Thus
30–40% of cell wall extension is irreversible. This indicates
that real plastic deformation exists in onion epidermal
cell walls. Using 200 or 500 mg for the pre-loading step as
well as 1 g for the loading did not qualitatively change this
cell wall behaviour. In all cases, a marked plastic component of the wall extension was detected (results not shown).
To explore the effect of net cellulose microfibril
orientation on plastic versus elastic components of cell wall
extension, longitudinal and transverse epidermal strips
were stretched in 1 min intervals by a series of equal loads
(2 or 5 g) up to a maximum of 8 and 20 g, respectively.
Thereafter, the loads were removed in 1 min intervals
back to the final 2 or 5 g stretching force. Thus the initial
2 or 5 g served as pre-loading steps in the experiments.
Such a spectrum of loads allowed estimation of the viscoelastic behaviour of onion cell walls subjected to a
maximally broad range of forces.
A typical experimental curve of a 2 g cycle is illustrated
in Fig. 6. In both epidermal strips, a significant part of
the wall deformation did not revert after unloading, which
confirmed its partly plastic nature. Statistical analysis of
many experimental curves of the type indicated in Fig. 6
has shown that loading induced much higher extensions in
transverse than in longitudinal epidermal strips (Fig. 7A)
and also that the elastic shrinkages during unloading were
higher to the same extent. Therefore, total deformation
reversibility (see Materials and methods) was statistically
the same for longitudinal and transverse epidermal strips
(Fig. 9A). Since the deformation at every step of loading
(unloading) includes a rapid initial deformation followed
2188 Suslov and Verbelen
Fig. 6. Extension of epidermal walls with net longitudinal (thick lines)
and transverse (thin lines) orientations of cellulose microfibrils in the
course of a 2 g loading/unloading cycle. Loads of 2 g were applied and
subsequently removed in 1 min intervals, and the resulting cell wall
deformation was recorded. Arrows indicate the time points at which the
loads were applied or removed. Numbers above the curves show the
total loads stretching the walls at the corresponding stages of the cycle.
Typical curves are shown.
by a time-dependent creep, the values of these components
were also analysed (Fig. 7B, C). The reversibility of
the initial deformation was similar (;60%) in longitudinal
and transverse strips (Fig. 9B). However, the two orientations differ specifically on the level of creep. On the one
hand, creep was significantly higher in transverse than in
longitudinal strips (Fig. 7C). On the other hand, the slow
time-dependent contraction upon unloading was similar in
both strips or even higher in longitudinal strips (Fig. 7C).
Therefore, the reversibility of creep in longitudinal strips
(14.2%) was significantly (P <0.01) higher than in transverse strips (8.9%). This means that the time-dependent
extension of onion epidermal walls is proportionally more
elastic parallel to the net cellulose orientation than perpendicular to the net cellulose orientation. The 5 g loading/
unloading cycles gave qualitatively similar results (Figs 8,
9). Longitudinal and transverse strips differed only in the
percentage of their creep reversibility, which was significantly higher in the former (6.5% compared with 3.4%)
(Fig. 9).
When comparing the two loading/unloading cycles, it is
clear that the total deformation (Fig. 9A) and its two
components (Fig. 9B, C) were much less reversible in the
5 g than in the 2 g cycle. Higher loads thus increase the
proportion of plastic deformation in onion epidermal walls.
Prolonged unloading steps (up to 15 min) revealed in
both cycles that the retarded elastic contractions described
in Figs 7C and 8C did not last for longer than 10 min
(results not shown). The dynamics and the amplitude of
the late part of the elastic contraction presented only a negligible fraction of the total deformation during unloading.
The effect of acid pH on cell wall extensibility
The orientation of cellulose microfibrils in the cell wall
seems to define the direction but not the rate of cell ex-
Fig. 7. Results of statistical analysis of cell wall extension curves of the
type indicated in Fig. 6 during 2 g loading/unloading cycles. Changes in
the total deformation (A), the initial deformation (B), and the creep (C)
of the epidermal walls with net longitudinal (black bars) and transverse
(white bars) orientation of cellulose microfibrils. Negative values indicate
contraction during unloading. The total deformation was calculated as
the change in wall length during 60 s between the subsequent load
applications and removals (see Fig. 6). The initial deformation is the
change in wall length measured 1 s after loading (unloading). The creep is
the amplitude of the time-dependent cell wall deformation during the
interval 1–60 s after load application (removal). Data are means 6SE
(n=21).
pansion (Baskin et al., 1999; Sugimoto et al., 2000). The
latter is probably regulated by specific cell wall proteins
breaking and/or establishing cross-links between different
cell wall polymers (McQueen-Mason et al., 1992; Fry
et al., 1992; Brownleader et al., 2000; Okamoto-Nakazato
et al., 2000). Among these proteins, the expansins induce
cell wall expansion at acid pH values by disrupting the
hydrogen bonding between cellulose microfibrils and
matrix hemicelluloses (Cosgrove, 2000b). It was hypothesized that different groups of factors regulate growth in
the directions parallel and transverse to the mean cellulose
orientation by catalysing shear and separation of microfibrils correspondingly (Baskin et al., 1999). To check
whether lowering the pH could stimulate extension differentially depending on the cellulose orientation, the effect
Cellulose orientation and wall mechanics
2189
Fig. 8. Results of statistical analysis of cell wall extension curves during
5 g loading/unloading cycles. Changes in the total deformation (A), the
initial deformation (B), and the creep (C) of epidermal walls with net
longitudinal (black bars) and transverse (white bars) orientation of
cellulose microfibrils. Negative values indicate contraction during
unloading. The total deformation was calculated as the change in wall
length during 60 s between the subsequent load applications and
removals. The initial deformation is the change in wall length measured
1 s after loading (unloading). The creep is the amplitude of the timedependent cell wall deformation during the interval 1–60 s after the load
application (removal). Data are means 6SE (n=10).
of an acid buffer on extensibility of transverse and longitudinal epidermal strips was studied.
Lowering the pH to 4.5 markedly (P <0.05) stimulated
the creep rate in longitudinal as well as in transverse
epidermal strips, but only during the time intervals 10–15
and 5–15 min after the load application (Fig. 10C, D). Its
effects on the initial deformations and the creep rates at the
earlier stages were not statistically significant (Fig. 10A and
B). The percentage of the creep rate stimulation (25–30%)
was the same for longitudinal and transverse strips (Fig.
10C, D). As a consequence, the ratio of their extensibilities
in the two directions is similar to that at neutral pH (Fig. 3).
Preincubation of the tissue in the acid buffer for 30 min
before stretching did not change the above-mentioned
pH effect on in vitro extensibility, which indicates that
Fig. 9. Reversibility (elasticity) of the total deformations (A), the initial
deformations (B), and the creeps (C) of epidermal cell walls with net
longitudinal (black bars) and transverse (white bars) orientation of
cellulose microfibrils in the 2 g and the 5 g loading/unloading cycles.
To estimate total deformation reversibility, the total deformations at all
steps of unloading-induced cell wall contraction were summed and
divided by the sum of the total deformation values at all steps of loadinginduced cell wall extension. The result was expressed as percentage
reversibility (elasticity). A similar procedure was used to calculate
separately the reversibility of creep and of initial deformation. Data
are means 6SE (n=21 for the 2 g loading/unloading cycle and n=10 for
the 5 g loading/unloading cycle).
2190 Suslov and Verbelen
Fig. 10. The effect of an acid pH buffer on the initial deformations (A) and the creep rates during the intervals 1 s–5 min (B), 5–10 min (C), and
10–15 min (D) of epidermal walls with net longitudinal and transverse orientation of cellulose microfibrils. Black bars, control treatment (10 mM sodium
citrate buffer, pH 6.0); white bars, experimental treatment (10 mM sodium citrate buffer, pH 4.5). Data are means 6SE (n=15).
tissue permeability was not a limiting factor (results not
shown). Thus the acid-induced extension of the isolated cell
walls does not depend on the mean cellulose microfibril
orientation.
Discussion
Upon loading, the epidermis samples stretched in a biphasic
way. The initial, practically instantaneous deformation was
followed by a slow time-dependent creep. In the past, both
parts of the extension have been considered as relevant wall
mechanical properties that could help to understand processes involved in cell expansion (Kutschera and Schopfer,
1986; Cosgrove, 1989). The fact that acid pH stimulated
the creep rate but did not increase the initial deformation
(Fig. 10) suggests that creep measured in vitro might be
a good indicator for the parameters U and Y that regulate
the rate of plant cell expansion in vivo and that the initial
deformation may characterize cell wall mechanical properties that have no relationship to in vivo growth regulation.
Similar conclusions were drawn in work on maize roots
(Büntemeyer et al., 1998).
For all loads used, the initial deformation and the creep
rate were higher perpendicular to the mean cellulose
orientation (transverse strips) than parallel to it (longitudinal strips) (Fig. 3). This confirms previous findings that
showed higher extensibility of onion epidermis walls in
the direction perpendicular to the mean microfibril orientation using a stress–strain method (Kerstens et al., 2001).
Okamoto and Okamoto (1994) have used the linear
dependence of the creep rate on the load to estimate /
and y, the in vitro analogues of U (yield coefficient) and
Y (yield treshold) parameters from the Lockhart equation.
In a similar approach for our data, linear fits were calculated for the ratio between late creep rates and loads. The
slopes of the lines obtained gave the values of /, which was
higher for transverse (0.41) than for longitudinal (0.36)
Cellulose orientation and wall mechanics
epidermal strips. This indicates that the mean orientation of
cellulose microfibrils has a moderate effect (14%) on /.
Loading/unloading experiments (Figs 5, 6) demonstrated the presence of a real plastic component in the
extension of onion epidermal cell walls in vitro. The plastic
component of time-dependent deformation is higher in
the direction transverse to the net microfibril orientation
than parallel to it (Fig. 9C). This difference is potentially
involved in growth anisotropy. Indeed, in vivo, a higher
proportion of the deformation could be transformed into
irreversible cell wall extension in the direction transverse
to the net cellulose orientation compared with the direction parallel to the net cellulose orientation. A real plastic
component has been described for rye coleoptiles (Kutschera, 1996), onion abaxial epidermis (Wilson et al.,
2000), and tomato fruit epidermis (Thompson, 2001). The
common methodological feature of all these studies was
the presence of a pre-loading step and consequently the
absence of a fully unloaded stage. However, Schopfer and
co-workers challenged the presence of an irreversible
plastic component in cell wall extension in vitro. They
came to the conclusion that apparent plastic deformation of
the wall in fact represented its retarded elastic behaviour
(Hohl and Schopfer, 1992; Nolte and Schopfer, 1997).
Their conclusion is based exclusively on experiments
where complete unloading took place. While being useful
in material testing, this step is physiologically irrelevant.
The creep test models the action of the turgor on the
growing cell wall. In this respect, complete unloading is
equivalent to the state of plasmolysis when the turgor
equals zero and plant cells do not grow. Cell expansion is
always associated with a state of stress inside the cell
wall. That is why measurements aimed at distinguishing
between reversible versus irreversible components of the
cell wall extension in vitro should be based on comparison
between different stress states of the cell wall. The magnitudes of these stresses should ideally be close to those
existing in the wall in vivo.
Cellulose and xyloglucans form the major load-bearing
network in the cell wall (Shedletzky et al., 1992) that
apparently plays a key role in determining the rate of the
wall extension during loading. At the same time, pectins
confer compression resistance to the cell wall (McCann
and Roberts, 1994). It is known that wall deformation
causes a certain microfibril realignment in the direction of
stress and a concomitant compression in the matrix (Spatz
et al., 1999; Köhler et al., 2000; Wilson et al., 2000). In the
onion cell walls, the pectin network, compressed by realigned microfibrils during loading, will then return to its
original shape after load removal. This process could
substantially contribute to the wall contraction observed
upon unloading. Thus the different amount of the retarded
elasticity correlating with the mean microfibril orientation
could result from the interaction between the two polymeric networks of the cell wall.
2191
Seemingly, a low pH, whose effect on the wall extension
might be mediated through expansin activation, is not
instrumental in defining the degree of wall mechanical
anisotropy. The stimulatory effect of a pH 4.5 buffer on the
creep rate of the epidermal walls was indeed similar parallel and transverse to their net microfibril orientation (Fig.
10C, D). Interestingly, isolated walls of Nitella were found
to be 10 times more sensitive to low pH in the longitudinal
(perpendicular to the microfibril orientation) than in the
transverse direction (Métraux and Taiz, 1979). This difference between a seed plant and an alga might be due to
the fact that expansins are absent in Characean algae that
have a different mechanism of acid-induced wall extension (Cosgrove, 2000a).
To sum up, this work provides direct evidence that the
net or mean predominant orientation of cellulose microfibrils confers to the cell wall of higher plants a mechanical
anisotropy that might be instrumental for growth anisotropy. Provided that extensibilities measured in vitro depend
on the U and Y parameters from the Lockhart equation, this
finding predicts that onion epidermal cells should grow
predominantly in the direction perpendicular to the mean
orientation of cellulose microfibrils in their cell walls. Not
only the higher extensibility but also the lower proportion
of retarded elasticity may contribute to the growth of a cell
in the direction perpendicular to the alignment of microfibrils in its cell wall. The validity of this hypothesis is
currently being studied on growing onion bulbs.
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