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Plant Physiol, September 2000, Vol. 124, pp. 397-406 The Mechanical Properties and Molecular Dynamics of Plant Cell Wall Polysaccharides Studied by Fourier-Transform Infrared Spectroscopy1 uráková,Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, United Kingdom
Polarized one- and two-dimensional infrared spectra were obtained from the epidermis of onion (Allium cepa) under hydrated and mechanically stressed conditions. By Fourier-transform infrared microspectroscopy, the orientation of macromolecules in single cell walls was determined. Cellulose and pectin exhibited little orientation in native epidermal cell walls, but when a mechanical stress was placed on the tissue these molecules showed distinct reorientation as the cells were elongated. When the stress was removed the tissue recovered slightly, but a relatively large plastic deformation remained. The plastic deformation was confirmed in microscopic images by retention of some elongation of cells within the tissue and by residual molecular orientation in the infrared spectra of the cell wall. Two-dimensional infrared spectroscopy was used to determine the nature of the interaction between the polysaccharide networks during deformation. The results provide evidence that cellulose and xyloglucan associate while pectin creates an independent network that exhibits different reorientation rates in the wet onion cell walls. The pectin chains respond faster to oscillation than the more rigid cellulose.
The cell wall is an elaborate
extracellular matrix that encloses each cell in a plant. It consists of
a microfibrillar cellulose phase and a matrix phase that contains a
variety of polymers such as poly-GalUA (PGA), hemicelluloses, proteins,
and phenolics, including lignin (Carpita and Gibeaut, 1993 The common onion (Allium cepa) has been used as a model
system for investigating the architecture of plant cell walls (McCann et al., 1992 With polarized light, the orientation of particular functional groups
can be determined with respect to the long axis of cells. Based on the
crystal structure analysis and polarized IR spectra, the tentative
assignment of native cellulose bands has been carried out (Cael et al.,
1975 Fourier-transformed IR (FT-IR) microspectroscopy has been used to
determine the presence and orientation of functional groups in
cellulose and pectin molecules within an individual plant cell wall
(McCann et al., 1993 To study the mechanical properties of the wall we have developed a
mechanical creep apparatus, which allows the displacement of the tissue
to be measured using a constant load applied axially to one end. The
tissue is mounted horizontally and the apparatus attached either to a
bright field microscope stage or to the stage of an IR microscope. The
hydration of the tissue can also be controlled using a specially
constructed cell (Chen et al., 1997 To investigate the nature, if any, of the interaction between the
macromolecular networks in stressed systems, we have used a new
technique called two-dimensional IR spectroscopy (2D FT-IR; Noda 1990
Effect of Hydration Cell walls are naturally composed of a hydrated polymer network,
and a knowledge of the influence of water and humidity is very
important in understanding the structure and role of particular cell
wall components. Edelmann (1995) To determine the water content in the samples, the water vapor
desorption at discrete relative air humidities (RH) was measured since
the equilibrium of hydration is generally described by water activity
(Aw). For a spectroscopic measure of water
content in the samples, the relative intensity ratio
(Ia) of the water deformation band at 1,640 cm Oriented Cell Wall Polysaccharides The typical parallel and perpendicular IR transmission spectra of
oriented onion epidermis in wet form together with their difference
spectrum (
In the spectra of the onion samples, backbone vibrations were assigned
for cellulose at 1,158 cm In the onion spectra, band shoulders from xyloglucan (XG), RG I, and
galactans can be present in the region at about 1,065 to 1,075 cm FT-IR Microspectroscopy During replicate analysis some variability in the IR spectra of the epidermis of different onions was noticed. This variability reflected differences in composition resulting from age, variety, storage conditions, etc. of the onion. However, we found that the compositional variability did not affect the main results of these experiments and that the polarization was not affected by this variability. Consequently, the results we have shown here are typical and illustrate reproducible findings related to the major biopolymer networks. In a typical epidermal cell in hydrated conditions at room temperature
before any stress is applied, the length and width of the cell were
approximately 125 and 40 µm, respectively (Fig. 3A). The spectra in Figure 3A were taken
from the epidermal cell at this initial stage, showing the parallel
(
Upon application of a 10-g load for 8 min the cell length increased to
175 µm and width decreased to 22 µm. This cell was chosen as it
showed the maximum change in dimensions although six other adjacent
cells increased in length by 32 to 50 µm and decreased in width by 7 to 18 µm. In the subtracted spectrum the PGA and cellulose
backbone vibrations gave strong positive bands at 1,110 and 1,018 cm Plastic and Elastic Deformation When the cell is allowed to relax after imposition of stress, the
dimensions of the length and width were 161 and 25 µm, respectively (Fig. 3C). Only partial recovery of cell dimensions in the original unstressed state was observed, and again the same six adjacent cells
decreased in length by 23 to 37 µm and increased in width by 10 to 15 µm. The subtraction spectrum showed little difference from that of
the stressed subtraction spectrum (Fig. 3B), although there was a
general small decrease in the absorbance intensity of the subtracted
relaxed spectrum in the 1,200 to 950 cm A large plastic deformation of the cell wall is confirmed in the subtraction spectrum of the relaxed cell (Fig. 3C), which shows a high degree of tissue orientation after the load has been removed. The small elastic deformation is shown by a slight reduction in the absorbance intensities of the subtraction spectrum of the relaxed cell (Fig. 3C) compared with the stressed cell (Fig. 3B). We can conclude that the stress-induced alignment for cellulose and non-cellulosic polysaccharide backbones is in the direction of stress with PGA side groups found orthogonal to this direction in a hydrated system. Under a constant stress, permanent deformation occurs in the walls and consequently in the cells themselves. 2D FT-IR Spectroscopy The FT-IR polarized microspectroscopy showed that the applied linear stress induces preferred macromolecular orientation in the cell walls parallel to the applied stress and the oriented macromolecules exhibited strong dichroism in certain bands. However, these results do not allow one to conclude whether the networks respond independently to applied stress or whether the PGA is forced into orientation through strong associative links with the cellulose. Therefore we used 2D FT-IR spectroscopy to look for evidence of an interaction between cellulose and non-cellulosic polysaccharides. The FT-IR spectroscopy revealed the variability of onion epidermis composition where variation in pectic polysaccharides and hemicellulose composition may occur in addition to changing relative amounts of PGA and cellulose. Spectra of bulk samples measured by dynamic 2D FT-IR tended to be very sensitive toward the composition variability. Because of the available transmission IR data for cellulose and PGA we took onion epidermis samples with high cellulose and PGA contents. Three repeat measurements of the onion samples showed that samples with comparable composition reveal good reproducibility of dynamic spectra. Dynamic 2D FT-IR Spectra Dynamic in-phase, quadrature, power, and phase spectra of a typical onion epidermis are shown in Figure 4. These spectra do not show the patterns of the normal transmission IR spectrum (Fig. 1); they only show responses of individual spectral bands to the applied strain, and a flat line where IR bands do not respond to the strain. In the wet onion tissue the 1,640 cm 1 band of adsorbed water was
not seen in the dynamic spectra, and neither the PGA ester bands (1,740 and 1,444 cm1) nor the carboxylate vibrations
of the pectate form (1,610 and 1,415 cm1) were
observed. Vibrational modes in the 1,800 to 1,200 cm1 region, mostly arising from side chains or
side groups (such as ester and OH), were apparently not very sensitive
to the applied perturbation. However, a significant response was found
in the bands related to the backbones of the polysaccharides in the
1,200 to 900 cm1 region. These are cellulose,
hemicelluloses, and pectic polysaccharide bands as listed in
Table I. However, it is not easy to unravel the XG, rhamnogalacturonan,
or galactan bands as they all contain a band or shoulder at about 1,070 cm1 and between 1,130 and 1,100 cm1
(Kauráková et al., 2000 ).
Parallel Polarization From the power spectrum it was apparent that the cellulose glycosidic stretching band at about 1,165 cm1
had a very high dynamic intensity in parallel polarization (Fig. 4A).
The negative band at 1,150 cm1 may contain some
XG and/or PGA contributions that have the same band position (Table I).
The PGA bands at 1,150; 1,110; and 1,006 cm1,
attributed to the backbone pyranoid ring vibrational modes, were of
medium intensity in positive orientation, whereas other bands at
1,056 and 1,029 cm1 were from cellulose in
negative orientation. The bands at about 1,075 cm1 can be attributed either to RG I, galactan,
or together with a band at 1,041 cm1 to XG
(Kauráková et al., 2000).
The cellulose band at 1,165 cm1 was positive in
both in-phase and quadrature spectra (Fig. 4B) with parallel polarized
light, as well as the band at 1,079 cm1. This
indicated that the glycosidic link and therefore the chain axis of
polysaccharides became more oriented in the direction of the applied strain.
The band at 1,040 cm1 assigned to XG was found
only in the in-phase spectrum. The predominantly negative
PGA-related bands at 1,103; 1,054; and 1,021 cm1 were stronger in the in-phase spectrum than
in the quadrature spectrum (Fig. 4B), whereas the cellulose was
more intense in the quadrature spectrum. This suggested a difference in
the response of the cell wall polymers to the applied stress. Bands in
the in-phase spectrum represent elastic response, whereas bands in the
quadrature spectrum indicate a more viscous behavior. The phase
spectrum of the onion epidermis is shown in Figure 4C. The phase angles
of the cellulose glycosidic link (1,165 cm1)
and a ring vibration probably from RG I or XG (1,079 cm1) were 7 and 8 degrees, respectively, higher
than the average. This corresponded to a response 2.0 ms slower than
the average phase delay. The phase angles of PGA vibrational modes at
1,152; 1,092; and 1,020 cm1 were about 2 degrees or 0.6 ms lower than the average. This may reflect the
spatially inhomogeneous composition of the bulk tissue.
Perpendicular Polarization The difference in the response of the polymers to the applied stress was found also in perpendicular polarization (Fig. 4D). No response from the glycosidic bonds at 1,165 to 1,150 cm1, but bands of higher intensity at 1,075 and
1,041 cm1 were found in positive orientation.
These bands may originate from RG I or XG and they were found in both
the in-phase and quadrature spectrum (Fig. 4D) with slightly higher
response to the applied strain in parallel orientation. The negative
bands (Fig. 4D) are ring vibrations from almost all the discussed
polysaccharides and their phase angles showed smaller values than in
parallel orientation (Fig. 4C). The 1,103 cm1
PGA band phase angle corresponded to 2.0 ms faster reaction than XG. No
phase angle different from the average was found for cellulose in the
perpendicular orientation. Because all the non-cellulosic polysaccharides except for PGA have bands about 1,075 cm1 (Table 1), further studies will be on model
compounds to distinguish them.
Two-Dimensional Correlation To decide whether the movements of cellulose and PGA are independent we examined the two-dimensional correlation spectra (Noda 1990; Budevska et al., 1994). Positive crosspeaks in the synchronous correlation spectrum showed intensity maxima of cellulose at 1,166 and
1,132 cm1 and the band at 1,079 cm1, which indicated that these modes responded
together to the applied strain in parallel polarized light. The
negative cross peaks corresponded to bands of PGA at 1,149; 1,114;
1,057; 1,030; and 965 cm1 (Fig.
5A). There were no crosspeaks between PGA
and cellulose peaks. Cellulose may interact with a polysaccharide at
1,079 cm1, however at the present state of
knowledge we are not able to prove whether this is also RG I or only
XG. The independent reorientational responses of cellulose and PGA
chain functional groups suggested that cellulose and PGA are not
directly associated.
1. At 1,057 and 1,021 cm1, negative PGA bands were found. Again there
were no cross-peaks between XG and PGA, excluding direct molecular interactions.
The observation of different rates with which the cellulose and PGA
networks react to the mechanical stress demonstrates the potential of
2D FT-IR to give new insight into the molecular interactions within
plant cell walls. To understand the significance, we have to consider
two findings from different techniques: (a) Mechanical tests performed
on whole plant and model tissues have shown that the different network
structures provide different mechanical properties. Cellulose dominated
the frequency response for small deformation (Whitney et al., 1999) and
(b) previous nuclear magnetic resonance measurements (Foster et
al., 1996; Ha et al., 1997) have shown a range of different molecular
mobilities in cell wall materials, with PGA molecules usually more
mobile then cellulose.
The 2D FT-IR data link these two above observations. When the tissue
was stretched, changes in cellulose IR bands dominated the power
spectrum. However, the phase angles showed that the cellulose response
was slower than the matrix, whereas some PGA chain reorientation
occurred faster. We conclude that the mechanical strain was taken up
first by the more mobile PGA matrix (including the middle lamella
between cells) and then by the cellulose microfibrils in the individual
cell walls. The role of XG and RG I remains open because of the
assignment difficulties.
FT-IR Microspectroscopy Biological Material For mechanical studies, strips of onion (Allium cepa) lower epidermis (15 mm long, 2 mm wide) from the equatorial section of the bulb scale were used. The first outer scale of the onion bulb was removed and discarded and subsequent scales were used. The lower epidermal strip was carefully removed from the onion scale with a forceps, then floated on distilled water for 30 min to allow any induced stress from the removal step to relax.Oriented Model Compounds Model compounds were used for assignment of polarization bands. Cotton cellulose fibers were manually co-aligned. A potassium pectinate film with 59% degree of esterification was prepared from aqueous solution at ambient temperature. The film was partially rehydrated and molecular orientation was introduced by stretching to about 10% of relative deformation ( l/l).
Mechanical Stress Studies The ends of the epidermal strip were glued onto stainless steel sample holders between the carriages in the creep apparatus, leaving an IR transparent sample area 8 mm long by 2 mm wide (Fig. 6). To control the humidity the sample was enclosed in a hydration cell fitted with two barium fluoride windows (1 mm thick, 13 mm in diameter), of which the upper one was held in a removable lid for sample loading (Chen et al., 1997). The
sample holders entered the cell through two small holes in the sides,
allowing free movement. Additional inlets and outlets allowed moist air
to be circulated using a peristaltic pump. Wet filter paper strips
inserted around the inner edge of the cell helped maintain a constant
humidity level around the tissue sample.
) of K2SO4 (97% RH),
Na2SO3 (95% RH), KBr (84% RH), and NaCl (76%
RH). After 48 h the samples were weighed and their IR spectra
collected. The samples were then air-dried at 35°C to obtain the dry
material content, and the water content was calculated from the
difference. The samples were measured only at high humidities because
onion shrinks and softens at 64% RH (Jones and Mann, 1963).
The creep apparatus allows the epidermal tissue sample to be stressed
while in a horizontal position. This is achieved by using a weight
attached to a moveable low friction carriage that is attached to one
end of the sample with the other end of the sample being fixed (Fig.
6).
FT-IR Microspectroscopy of Stressed Samples The IR spectra of stressed samples were measured with a spectrometer (FTS 175C, Bio-Rad, Cambridge, MA) with an external microscope (UMA 500, Bio-Rad) attachment that allows detailed measurements of a single cell down to an area of 10 × 10 µm, with a polarizer in the optical port and an adjustable aperture at the remote focal plane. A KRS5 (thallium bromide and iodide mixed crystal) wire grid polarizer (Graseby Specac Ltd., Orpington, Kent, UK) was used to polarize the IR beam. Spectra were measured with the incident light polarized parallel ( )
and perpendicular ( ) to the stretching direction. Subtraction spectra were calculated as parallel minus perpendicular polarized spectrum (-).
In preliminary experiments we found that a water content of the sample
of about 70% (w/w) was optimal for spectroscopic measurements and this
was used in all subsequent experiments.
A sample area in the center of one of the lower epidermal cells
(typically 10 × 50 µm) was selected for collecting the IR spectra. A pair of single-beam spectra in parallel and perpendicular polarizations were collected from the same spot on the cell being sampled. For each sample a background spectrum without sample in place
was obtained with the same polarization and humidity conditions. The
spectral resolution used was 8 cm1 and 128 interferograms
were co-added to improve spectral quality. This procedure was carried
out for the same cell with the initial, stressed, and relaxed
conditions as described below.
The parallel and perpendicular spectra of the epidermal layer were
obtained from the onion lower epidermis in the relaxed condition. A
load of 10 g was then applied for a fixed time period, which
stressed the sample, and the displacement was measured as a function of
time. Finally the weight was removed and the sample was allowed to
relax for a fixed time accompanied by logging of the displacement. The
cell images and spectra in both polarization directions were acquired
for each of these steps.
The cell images were acquired with a camera (CCD-IRIS/GGB, Sony, Tokyo)
attached to the microscope and processed with a video capture card in a
PC using Shadow II software (Bio-Rad).
FT-IR Spectroscopy of Model Compounds Polarised FT-IR spectra of oriented models were measured on a spectrometer (FTS 6000, Bio-Rad) with a KRS5 wire grid polarizer. The dry cotton cellulose fibers were co-aligned and pressed in a diamond anvil cell (Diacell, Leicester, UK). The potassium pectinate film was prestretched while kept in the humid hydration compartment and measured in the polymer stretcher sample holder.2D FT-IR Spectroscopy For dynamic 2D FT-IR spectral measurements an epidermal strip
(1 × 1.5 cm) was placed in a polymer stretcher (Bio-Rad), which was modified to enclose the sample in a hydration cell with barium fluoride windows based on the design of Chen et al. (1997) The 2D FT-IR measurements were carried out on a spectrometer (FTS 6000, Bio-Rad) in step scan mode using a narrow band nitrogen cooled MCT
(HgCdTe) detector. A KRS5 wire grid polarizer was used to obtain
radiation polarized parallel and perpendicular to the stretching
direction, which was chosen to coincide with the axis of the cell wall
elongation. Four scans at 8 cm Two-dimensional spectra were obtained from the modulated step-scan spectra using Digital Signal Processing. The phase modulation frequency was 400 Hz. The in-phase static spectra were normalized against a single beam background spectrum and the in-phase and quadrature dynamic spectra were normalized against the in-phase static spectrum. The phase spectra were calculated with the spectrometer software (Bio-Rad) and the power spectra were calculated in Matlab (Mathworks, Inc., Natick, MA). 2D FT-IR Spectra Dynamic IR spectroscopy can be used to examine individual
submolecular constituents (e.g. backbone segments, side chains, and
various functional groups) in a polymer system where the variation of
IR dichroism is measured as function of time. Dichroisim is the
property of molecules such that the IR absorption bands measured with
light polarized parallel and perpendicular to any reference direction
are not identical and infers the presence of orientation at the
molecular level. The response of side chain functional groups may not
be fully synchronized with polymer backbone motions (Noda et al.,
1999 Two-dimensional cross-correlation of the dynamic spectral response at
each wavelength is used to infer the existence of connectivity and
interactions of functional groups. In synchronous plots the autocorrelation bands lie on the diagonal and off-diagonal crosspeaks exist between bands with similar time response (Noda, 1990 A phase spectrum provides for each absorption band the phase delay to
the stretching frequency
The authors thank Dr. M.C. Jarvis and Dr. M.C. McCann for comments on the manuscript.
Received January 1, 2000; accepted May 24, 2000. 1 M.K. (on leave from SAS, Bratislava, SK) was supported by a NATO Royal Society fellowship and R.H.W., A.C.S., P.K.S., N.W., and K.W.W. were suported by a Biotechnology and Biological Science Research Council Competitive Strategy Grant.
* Corresponding author; e-mail reg.wilson{at}bbsrc.ac.uk; fax 44-1603-507723.
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ABSTRACT
INTRODUCTION
= 20 Hz and a sample
modulation amplitude of 200 µm.
