Cellulose (2014) 21:1075–1086

DOI 10.1007/s10570-013-0121-2

ORIGINAL PAPER

Cellulose microfibril orientation in onion (Allium cepa L.)

epidermis studied by atomic force microscopy (AFM)
and vibrational sum frequency generation (SFG)
spectroscopy
Kabindra Kafle • Xiaoning Xi • Christopher M. Lee •

Bernhard R. Tittmann • Daniel J. Cosgrove •

Yong Bum Park • Seong H. Kim

Received: 9 July 2013 / Accepted: 19 November 2013 / Published online: 3 December 2013
Ó Springer Science+Business Media Dordrecht 2013

Abstract Cellulose microfibril orientation in plant
cell walls changes during cell expansion and devel-
opment. The cellulose microfibril orientation in the
abaxial epidermis of onion scales was studied by
atomic force microscopy (AFM) and sum frequency
generation (SFG) vibrational spectroscopy. Onion
epidermal cells in all scales are elongated along the
onion bulb axis. AFM images showed that cellulose
microfibrils exposed at the innermost surface of the
abaxial epidermis are oriented perpendicular to the
bulb axis in the outer scales and more dispersed in
the inner scales of onion bulb. SFG analyses can
Electronic supplementary material The online version of
this article (doi:10.1007/s10570-013-0121-2) contains supple-
mentary material, which is available to authorized users.
K. Kafle  C. M. Lee  S. H. Kim (&)
Department of Chemical Engineering and Materials
Research Institute, The Pennsylvania State University,
University Park, PA 16802, USA
e-mail: shkim@engr.psu.edu
X. Xi  B. R. Tittmann
Department of Engineering Science and Mechanics,
The Pennsylvania State University, University Park,
PA 16802, USA
D. J. Cosgrove  Y. B. Park (&)
208 Mueller Laboratory, Department of Biology,
The Pennsylvania State University, University Park,
PA 16802, USA
e-mail: yzp107@psu.edu
determine the orientation of cellulose microfibrils
averaged over the entire thickness of the cell wall. We
found that the average orientation of cellulose micro-
fibrils inside onion abaxial epidermal cell walls as
revealed by SFG is similar to the orientation observed
at the innermost cell wall surface by AFM. The
capability to determine the average orientation of
cellulose microfibrils in intact cell walls will be useful
to study how cellulose microfibril orientation is related
to biomechanical properties and the growth mecha-
nism of plant cell walls.
Keywords Onion epidermis  Cellulose
microfibril  Microfibril orientation  Sum
frequency generation spectroscopy  Atomic
force microscopy
Introduction
Cellulose microfibril orientation in plant cell walls is
considered to be an important determinant of wall
biomechanics and growth of plant cells (Cosgrove
2000; Baskin 2005). Several models have been
proposed for the role of cellulose microfibril orienta-
tion in actively growing plant tissues (Roelofsen and
Houwink 1953; Roland et al. 1977; Green 1964;
Neville et al. 1976). It is known that the movement of
cellulose synthase complexes (CSCs) located in the
underlying plasma membrane determines the initial
orientation of cellulose microfibrils that are deposited
at the innermost surface of the cell wall (Giddings Jr

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and Staehelin 1988; Heath 1974). The CSC movement
is related to the orientation of the underlying cellular
microtubule cytoskeleton (Paredez et al. 2006; Li and
Gu 2012). However, the factors regulating the orien-
tation of cellulose microfibrils in growing plant walls
are not fully understood yet (Baskin 2005; Sugimoto
et al. 2000; Richmond et al. 1980; Preston 1974;
Kutschera 2008; Neville 1985). According to the
multinet growth hypothesis (Roelofsen and Houwink
1953; Green 1960), cellulose microfibrils are depos-
ited along the transverse direction with respect to cell
elongation and then passively reoriented along the
longitudinal direction during cell elongation (Baskin
2005). Such passive reorientation has been observed in
actively growing Arabidopsis roots (Anderson et al.
2010) but was not observed in in vitro extension
studies in cucumber hypocotyls (Marga et al. 2005).
The cellulose microfibril deposition and reorientation
behaviors in intact plant cell walls could be studied
with analytical techniques that can selectively probe
the cellulose microfibrils in intact cell walls. These
techniques can also provide critical insights into
understanding the biomechanics and elongation of
plant cell walls at various developmental stages
(Kutschera 2008).
High-resolution electron microscopy has been
widely used for direct visualization of cellulose
microfibrils in onion parenchyma cell walls after fast
freeze/deep etch followed by metal deposition (McC-
ann et al. 1990; Satiat-Jeunemaifre et al. 1992; Wells
et al. 1994). In electron microscopic imaging, fixative
and staining agents were used to prepare the cell wall
samples. Removal of noncellulosic polysaccharides
was often required for high-resolution imaging of
primary cell walls (Davies and Harris 2003). However,
pretreatment of cell wall samples might alter the
native arrangement of microfibrils (McCann et al.
1990). Recently, the organization of cellulose micro-
fibrils has been visualized by atomic force microscopy
(AFM) for celery parenchyma (Thimm et al. 2000),
cucumber hypocotyls (Marga et al. 2005), maize
primary cell walls (Ding and Himmel 2006), and
onion epidermis (Zhang et al. 2013). The AFM
imaging technique allows visualization of cellulose
microfibrils exposed at the cell wall surface. However,
the average orientation through the entire cell wall
cannot be visualized through surface imaging only.
Fourier-transform infrared (FTIR) spectroscopy
with a polarized-light source has been used to measure
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Cellulose (2014) 21:1075–1086
the absorbance of specific vibration modes parallel
and perpendicular to the anisotropic growth axis of the
cell to determine the net orientation of cellulose
microfibrils inside cell walls (Chen et al. 1997).
However, this method could suffer from spectral
interferences from other, noncellulosic matrix poly-
mers in the cell wall. Polarized-light imaging can be
used to determine the average orientation of Congo
Red dye adsorbed onto cellulose with its molecular
axis parallel to the cellulose chain (Suslov et al. 2009).
However, Congo Red also binds to xyloglucan (Wood
1980; Wood et al. 1983), which can complicate the
interpretation of such results.
Study of cellulose microfibril orientation could
benefit from a nondestructive and noninvasive spec-
troscopic characterization method that can distinguish
cellulose from noncellulosic components in plant cell
walls. The noncentrosymmetry requirement of non-
linear optical spectroscopy, such as sum frequency
generation (SFG) vibrational spectroscopy, makes
differentiation between cellulose and noncellulosic
components possible (Barnette et al. 2011). Selective
detection and quantification of crystalline cellulose by
SFG has been demonstrated for intact woody cell
walls (Barnette et al. 2011, 2012). Although SFG is a
well-known surface-sensitive analytical technique, the
SFG response of cellulose is dominated by the signal
from the bulk crystal (Lee et al. 2013a).
In SFG, two high-intensity laser pulses with
infrared and visible frequencies are temporally and
spatially overlapped on the sample. When the fre-
quency of the infrared beam resonates with the
vibration modes that are arranged without centrosym-
metry in the sample, a new photon whose frequency is
the sum of the two input frequencies ðxSFG ¼ xVis þ
xIR Þ can be generated. The intensity of the SFG signal
is proportional to the square of the second-order
nonlinear susceptibility of the vibration mode (Lam-
bert et al. 2005). This second-order nonlinear suscep-
tibility is zero for a noncrystalline or centrosymmetric
medium. Thus, disordered polysaccharides such as the
wall matrix components cannot generate SFG signals.
Cellulose has noncentrosymmetrically arranged func-
tional groups in its crystal structure and can thus have
nonzero values for the second-order nonlinear sus-
ceptibility. The magnitude of this term is highly
sensitive to the density and preferential alignment of
crystalline cellulose microfibrils within the optical
coherence range of the SFG process (LaComb et al.
Cellulose (2014) 21:1075–1086
2008). When the SFG photons are detected in a reflection
geometry, the SFG coherence length along the direction
normal to the surface is expected to be on the order of
hundreds of nanometers (LaComb et al. 2008).
In cellulose I crystals, the methine (CH) groups at the
axial position of the glucopyranose ring are positioned on
the opposite sides of the planar sheet: (200) in
cellulose Ib and (110) in cellulose Ia (Zugenmaier
2008). The oppositely pointing transition dipoles of the
methine stretch vibration cancel each other, making
them SFG inactive. Thus, the methine peak at
*2,900 cm-1 is absent from cellulose SFG spectra,
although it is a dominant peak in infrared (IR) and
Raman spectra (Barnette et al. 2011; Lee et al. 2013b;
Wiley and Atalla 1987). In contrast, the exocyclic CH2
groups and intrachain hydrogen-bonded OH groups
point in the same direction within the cellulose I crystal;
thus, their dipoles add up, forming net dipoles across the
entire cellulose crystal (Lee et al. 2013a). This polar
ordering of the CH2 and OH groups in the cellulose I
crystal meets the noncentrosymmetry requirement on the
nanoscale. In the case of fully grown secondary cell walls
such as those of woody cells, the sharp CH2 vibration
peak appears at 2,944 cm-1 and the intrachain hydro-
gen-bonded OH vibrations are centered at *3,320 cm-1
(Barnette et al. 2011; Lee et al. 2013a, b).
Preferential alignment of these polar crystallites
within the optical coherence length scale is critical for
SFG. The SFG intensity of a specific vibration mode
depends on the angle between the direction of net
transition dipole in the sample and the electric field
vectors of the laser beams (Wang et al. 2005). When
two SFG-active vibration modes are directed at
Fig. 1 White onion bulb
showing scales with
annotation (1st: outer, older
scale; 11th: inner, younger
scale). Schematic shows
abaxial and adaxial
epidermis in one onion scale
(cartoon schematic not to
scale)
1077
different angles, their relative intensities can provide
the net orientation of the crystallites. When the electric
vector of the IR beam is aligned with the transition
dipole of the vibration mode, the SFG intensity of that
vibration mode can be enhanced (Hieu et al. 2011). In
cellulose, the O3–H and O2–H hydroxyl groups are
involved in intrachain hydrogen bonding with oxygen
O5 and O6 in the adjacent glucose unit, respectively
(Marechal and Chanzy 2000). Thus, the O3–HO5
and O2–HO6 groups are aligned along the cellulose
chain axis (Lee et al. 2013b). In contrast, the stretch
mode of the exocyclic CH2 groups has its vibration
dipole moment directed off from the chain axis (Wiley
and Atalla 1987; Lee et al. 2013b). Thus, the relative
intensity of these SFG peaks can provide the cellulose
microfibril orientation in the sample.
In this study, we employed SFG spectroscopy and
AFM imaging to investigate the net orientation of
cellulose microfibrils in the abaxial epidermal cell
wall of onion scales. Onion was chosen since it has
been studied widely as a model for primary cell walls
(McCann et al. 1990; Suslov et al. 2009; Ha et al.
1997; Wilson et al. 2000; Ng et al. 2000; Mita and
Shibaoka 1983). The outer and inner scales of onion
could represent various developing stages of primary
cell walls, with inner scales being younger and outer
scales being older. Thus, onion scales provide an
opportunity to investigate cellulose microfibril orien-
tations at various developmental stages. A single
onion bulb scale is composed of three distinct parts:
abaxial epidermis at the convex side (away from the
bulb axis), fleshy parenchyma tissue in the middle, and
adaxial epidermis at the concave side (facing the bulb
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axis) (Fig. 1). The abaxial epidermis cell is split open
along the sidewalls when the skin is peeled from each
bulb scale (schematic in Fig. 1). Thus, the peeled skin
is the outer wall of the epidermal cell and the
cytoplasm can be washed off. The inner side of the
peeled skin exposes the newly deposited cellulose
microfibrils (Green 1962). Thus, AFM could be
readily used to image cellulose microfibrils of such
inner cell wall surface (Zhang et al. 2013). Also, the
peeled abaxial epidermal walls are thinner than the
probe depth of the SFG process; thus, the average
orientation of microfibrils inside the cell wall can be
determined by the polarization dependence of SFG.
Materials and methods
Preparation of cell walls of abaxial epidermis
of onion scale
White onion (Allium cepa L. cometa) bulbs (bulb
diameter 8–10 cm) were obtained from a local
grocery, and the outermost dry scales were removed.
Fig. 2 Optical microscopy images of cell walls from onion
abaxial outer epidermis of a 2nd layer, b 5th layer, c 8th layer,
and d 11th layer (scale bar 200 lm). In b, two-headed arrows
indicate the longitudinal (dashed line) and transverse (solid line)
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Cellulose (2014) 21:1075–1086
The first fresh scale from the outside was numbered 1,
and higher numbers were assigned to subsequent inner
scales. Onion bulbs used in this study typically
contained 11 or 12 scales. Abaxial epidermal layers
from the 2nd, 5th, 8th, and 11th scales were carefully
peeled (Fig. 1) and mounted on a glass slide with the
cell wall surface close to the plasma membrane facing
up, following the procedure of Zhang et al. (2013).
Epidermal cell sizes were measured from optical
images taken with an inverted transmitted-light
microscope (Fig. 2). In all epidermal images, cell
nuclei were not seen, which confirmed that the peeled
layer contained only the outer half of the epidermal
cell. Image analysis software (Scion, NIH) was used to
measure cell length and width. The statistical average
of cell size was obtained from optical images of
43–174 cells from three onion bulbs, depending on the
cell size variances in each scale. Cell size increases
from the inner scale to the outer scale. In the 11th scale
images, several cells showed division planes (marked
with arrows in Fig. 2d, inset). Table 1 summarizes the
average cell size measured for each abaxial epidermal
layer with the standard error of the mean (SEM).
directions with respect to the cell elongation axis. The
longitudinal direction is along the onion bulb axis. The inset
to d shows walls of recently divided cells (arrows)
Cellulose (2014) 21:1075–1086 1079

Table 1 Cell size measurements from four abaxial epidermal layers

Onion scale no. Total number of Longitudinal (lm) Transverse (lm) Aspect ratio
cells measured (n) AVG ± SEM AVG ± SEM AVG ± SEM

2 47 336 ± 17 70 ± 2 5.2 ± 0.4

5 81 206 ± 7 85 ± 2 2.6 ± 0.1
8 150 178 ± 4 55.5 ± 0.8 3.4 ± 0.1
11 174 67 ± 2 23.5 ± 0.2 2.9 ± 0.1

Table 2 Cellulose content and wall thickness measured by
profilometry (air-dried cell walls)
Abaxial Cellulose content Air-dried cell
epidermis in alcohol-insoluble wall thickness
from onion residue (%) (lm)
scale no. AVG ± SEM AVG ± SEM
(n = 4) (n = 25)
2 36.6 ± 1.4 4.9 ± 0.1
5 31.3 ± 1.4 3.4 ± 0.1
8 28.2 ± 1.1 3.2 ± 0.2
11 27.0 ± 0.9 1.7 ± 0.1
Cellulose content and wall thickness of abaxial
epidermal cell walls
The cellulose content in the abaxial cell wall was
estimated by the phenol–sulfuric acid method (Dubois
et al. 1956). Epidermal layers from each scale were
collected from several white onion bulbs, pulverized
into powder in liquid nitrogen, and lyophilized. For
each sample, 5 mg alcohol-insoluble residue was
treated three times with 20 mM cyclohexane diamine
tetraacetic acid (CDTA, pH 6.5) at room temperature
(RT) overnight, followed by 39 treatments with
50 mM Na2CO3 containing 20 mM NaBH4 at RT
overnight, incubation in 4 M NaOH containing
20 mM NaBH4 at RT overnight (with three
exchanges), washed with distilled-deionized water
(ddH2O) until neutralized, and then lyophilized. The
4 M NaOH-insoluble residues were hydrolyzed by
72 % H2SO4 at RT for 1 h, and then diluted with
ddH2O to 2 M H2SO4 and incubated at 100 °C with
frequent vortexing (every 10 min) until the solid
matter was fully dissolved. The sugar amounts
digested by H2SO4 were pooled and estimated by the
phenol–sulfuric acid method (Dubois et al. 1956) to
provide an estimate of cellulose content. Table 2
compares the cellulose content in each cell wall.
The cell wall thickness of onion layers air-dried on
a glass slide was measured by optical profilometry
(Zygo NewView 7300). For each epidermal layer, 25
cells from three different onions were imaged, and the
average cell wall thickness is presented in Table 2.
AFM measurements
Onion epidermis samples were prepared for AFM
imaging as described previously (Zhang et al. 2013).
Freshly peeled cell walls were fixed onto glass slides
with double-sided tape. The epidermal strips were then
immersed in phosphate-buffered saline solution (pH
7.4) containing 0.1 % Tween-20 for 1 h. After that,
the samples were rinsed with distilled water. Epider-
mal wall strips were scanned by AFM (Veeco
Dimension ICON from Bruker) in Peak Force QNM
in fluid (maximum force 1–3 nN; scan rate 1 Hz).
Nanoscope (v 8.10b44) was used to control AFM
operation, and Nanoscope Analysis software (v 1.40)
was employed for image processing. A cantilever
holder from Bruker for liquid imaging was used to
keep samples fully hydrated in water during the scan.
The AFM tips used for imaging were ScanAsyst
Fluid? probes from Bruker. The AFM cantilever
normal spring constant was *0.7 N/m. The spring
constant of each cantilever was calibrated using a
thermal tuning method before experiments (Hutter and
Bechhoefer 1993). All images were generated by
scanning in the direction along the longitudinal
direction of the cells (Fig. 3). Five representative
topography images for each scale from five different
onion bulbs were chosen for microfibril angular
distribution analysis. Each image was overlaid with
a 10 9 10 mesh grid, and at each mesh point, the angle
of either intersecting or the nearest microfibril was
measured with respect to the transverse axis of the cell.
Zero degrees was defined as the microfibril angle
along the transverse axis of the cell. In this way, a total

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1080
Fig. 3 a Schematic view of the AFM tip imaging the inner
surface of the epidermal cell wall under water. b The AFM scan
direction along the longitudinal axis of the cell (parallel to the
bulb axis)
of 500 microfibril angles were measured for each
scale.
Polarized FTIR measurements
To compare with the conventional analysis method
(Chen et al. 1997), infrared spectra were collected
using a Nicolet 8700 FTIR spectrometer (Thermo
Scientific), a KBr beam splitter, and a deuterated
triglycine sulfate (DTGS) detector. A strip of dry
onion layer was attached on a 3 mm 9 3 mm hole of a
sample holder. An IR wire grid polarizer (Harrick
Scientific Products, PWG-U1R) was used to produce a
polarized infrared light source. IR spectra were taken
with the IR polarization axis along the longitudinal
and transverse directions of the sample. A background
spectrum was taken before each polarization spec-
trum. A total of 200 interferograms were averaged
with spectral resolution of 4 cm-1.
SFG spectroscopy measurements
Air-dried onion epidermal layers were analyzed by
vibrational SFG spectroscopy. Details of the SFG
system (EKSPLA) were described previously (Bar-
nette et al. 2011). Briefly, it consisted of a picosecond
Nd:YAG 1,064-nm laser operated at 10 Hz repetition.
A 532-nm visible laser pulse was generated through
frequency doubling of the Nd:YAG output. A tunable
2.3–10-lm infrared laser pulse was generated through
optical parameter generation and amplification pro-
cesses. The p-polarized infrared beam and s-polarized
visible beam were temporally and spatially overlapped
at the onion epidermal layer at incidence angles of 56°
and 60° with respect to the surface normal, respec-
tively. The p- and s-polarizations are defined as the
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Cellulose (2014) 21:1075–1086
electric field of the light within and out of the laser
incidence plane, respectively. The SFG signal was
detected in the reflection geometry with a beam
collimator to increase the signal collection efficiency.
The s-polarized SFG signal was filtered through a
monochromator and recorded with a photomultiplier
tube. SFG spectra were taken at a 4 cm-1 interval in
the C–H stretch vibration region (2,700–3,050 cm-1)
and an 8 cm-1 interval in the O–H stretch vibration
region (3,100–3,800 cm-1). The SFG signal was
averaged over 100 laser pulses and normalized by
the IR and visible input laser intensities at each shot.
Due to the local heterogeneity of the onion epidermis
sample, individual SFG spectra from ten different
locations were averaged to create the most representa-
tive spectra for each sample. The SFG spectra shown in
this paper are the averaged ones. The probe volume of
our SFG system was estimated to be about
200 lm 9 150 lm wide over the sample and approx-
imately 20 lm deep from the external surface of the
sample (Figs. S1 and S2 in the Electronic Supplemen-
tary Material). Since the onion epidermal cell walls
were much thinner than this probe depth, the SFG signal
from the epidermal cell wall was the average over the
entire thickness of the wall. To study the polarization
dependence of the SFG signal of each layer, spectra
were recorded with the laser incidence plane along the
longitudinal and transverse directions of the cell as
shown in Fig. 2. The peak areas of CH2 and OH stretch
regions reported in this paper were calculated by
integrating the major peaks centered at 2,920 cm-1
(mCH2 ) and 3,320 cm-1 (mOH), respectively.
Results and discussion
Surface cellulose microfibril orientation assessed
by AFM
Figure 4 shows AFM topography images of the inner
surface of the abaxial epidermal layers. The bright-
contrast lines in the images represent cellulose
microfibrils, possibly coated with pectins and xylo-
glucan (Davies and Harris 2003; Zhang et al. 2013).
Preferential orientation of the microfibrils along the
transverse direction was observed in the epidermis of
the 2nd layer (Fig. 4a), whereas the cellulose micro-
fibrils were found to be less oriented in the 11th layer
(Fig. 4d). The orientation distribution plots clearly
Cellulose (2014) 21:1075–1086 1081

Fig. 4 Cellulose
microfibril orientation in the
plasma-membrane-side cell
wall of the abaxial epidermis
of onion scales. AFM
images of hydrated onion
abaxial epidermis cell wall
from the a 2nd layer, b 5th
layer, c 8th layer, and d 11th
layer (scale bar 0.5 lm).
The insets show the angular
distribution of the cellulose
microfibrils. The transverse
direction (horizontal
direction in the figure) is set
to zero degrees in the
histogram

show a progressive change in the microfibril orienta-
tion anisotropy. The microfibrils were highly oriented
along the transverse direction in the second layer
(Fig. 4a). The microfibrils in the fifth layer spanned a
slightly broader angular distribution (Fig. 4b). The
preferential orientation along the transverse direction
was reduced in the eighth layer (Fig. 4c). In the 11th
layer, the microfibril angle varied over a wide range
without specific directionality (Fig. 4d).
Average orientation measured by polarized FTIR
Since AFM can detect only microfibrils exposed at the
cell wall surface, the question of whether microfibrils
inside the cell wall have the same orientation or not
must be examined independently. Polarized transmis-
sion FTIR analysis has been used to probe the average
orientation across the entire thickness of the cell wall
(Chen et al. 1997). The transmission FTIR spectra
with IR beams polarized along the longitudinal and
transverse axes of the cell are shown in Fig. 5. In the
C–O and C–C stretch region (950–1,250 cm-1) of the
second-layer spectrum, the peak intensities at 1,018,
1,066, 1,103, and 1,153 cm-1 were slightly stronger
when the IR polarization was parallel to the transverse
direction (red line in Fig. 5) compared with the
longitudinal direction (black line in Fig. 5). Since
noncellulosic matrix polymers (such as pectins and
hemicelluloses) can also contribute peaks in this
fingerprint region (Wilson et al. 2000; Wellner et al.
1998), the difference between the two polarizations
was not drastic. Moreover, it has been reported that
pectins could be aligned along the cellulose microfi-
brils (Yoneda et al. 2010). Although such overlaps and
closeness in IR absorption bands may pose ambiguity
in determining the accurate orientation of cellulose
microfibrils in intact cell wall samples, the data shown
in Fig. 5 suggest that there is anisotropy in the
microfibril orientation in the outer layers.

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Fig. 5 FTIR transmission spectra of onion abaxial layers
collected with the IR polarization along the longitudinal (black)
and transverse (red) directions. (Color figure online)
Net cellulose microfibril orientation measured
by SFG
The ambiguity in assigning FTIR fingerprint bands can
be avoided in SFG analysis since SFG selectively
detects crystalline cellulose inside the sample (Barnette
et al. 2011). The SFG spectra of the abaxial epidermal
layers collected with the laser incidence plane aligned
along the longitudinal and transverse axes of the
elongated cell are shown in Fig. 6. The overall SFG
signal intensity decreased from the outer to inner
scales. This can be attributed to the decrease in the
thickness and cellulose content of the cell wall
(Table 2). The peak at 3,320 cm-1 in the OH stretch
region is consistent with the SFG spectra observed for
secondary cell walls (woody cells and flax fibers) as
well as Avicel cellulose (Barnette et al. 2011; Lee et al.
2013a). The shoulder peak at 3,450 cm-1 is much more
prominent than that observed for woody cell walls
(Barnette et al. 2011). This peak was also observed in
SFG for the secondary cell walls of Arabidopsis (Park
et al. 2013) as well as in FTIR from spruce wood
secondary cell walls (Fernandes et al. 2011) and celery
collenchyma primary cell walls (Thomas et al. 2013).
In previous IR studies, the peak near 3,450 cm-1 was
assigned to the hydroxyl groups at the microfibril
surface accessible to water (Fernandes et al. 2011;
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Cellulose (2014) 21:1075–1086
Thomas et al. 2013). The peak positions of these
hydroxyl groups would be sensitive to interactions
between the cellulose chains at the crystal surface and
surrounding hemicelluloses or pectins. The
3,450 cm-1 peak is negligible for pure microcrystalline
cellulose (Barnette et al. 2011), flax fibers (Lee et al.
2013a), and pure cellulose Ia and Ib isolated from
Glaucocystis and Halothyncia (Lee et al. 2013b).
Another difference from the cellulose SFG spectra of
secondary cell walls is that the CH2 asymmetric stretch
region is broad and centered at *2,920 cm-1 with a
shoulder peak at *2,964 cm-1 for the second, fifth,
and eight layers. The peak center and width of the CH2
SFG peak of the second, fifth, and eighth onion
epidermis cell walls are similar to those observed for
the primary cell walls of Arabidopsis (Park et al. 2013).
These onion cell walls also have the characteristics of
primary cell walls; i.e., the cells are still growing, and
the cell walls do not contain any lignin. It is noted that
the weak CH2 SFG peak of the innermost 11th layer is
centered at 2,944 cm-1. The origin of this difference is
not clear yet, but it may be due to the difference in the
cellulose microfibril ordering and bundling. Further
details will be the subject of future work.
In Fig. 6, we note that the relative intensities of the
CH2 and OH peaks change greatly when the direction
of the laser incidence plane changes from longitudinal
to transverse. In previous SFG study of inflorescence
stems of Arabidopsis, the CH2/OH intensity ratio
changed in a similar way along the maturation and
development gradient (Park et al. 2013). This raises
the question of whether changes in cellulose orienta-
tion along the Arabidopsis inflorescence stem might
be the basis for the axial gradient in CH2/OH intensity
ratio. We deem this unlikely because the Arabidopsis
samples were composed of finely ground cell walls
where the sample volume included many wall frag-
ments in randomized orientation. In that study the
change in the CH2/OH intensity ratio was attributed to
alteration in lateral ordering or packing of cellulose
along the stem axis. Thus, the CH2/OH intensity ratio
can be influenced by multiple features of cellulose
organization (both orientation relative to the laser
beam and packing order).
The ratios of the CH2 and OH peak areas for the
longitudinal and transverse SFG spectra are plotted in
Fig. 7. The CH2/OH ratio is significantly smaller in the
transverse direction compared with the longitudinal
direction; the exception is the epidermal layer of the
Cellulose (2014) 21:1075–1086 1083

Fig. 6 SFG spectra of

abaxial epidermal layers
from four different scales.
The polarization
combination was s for the
SFG signal, s for visible, and
p for IR. The laser incidence
plane was aligned along the
a longitudinal and
b transverse directions of the
cell. Inset diagrams above
the spectra show the onion
epidermis strip orientation
with respect to the laser
incidence plane in each case

11th scale. In the second and fifth scales, the OH

intensity increases significantly in the transverse
direction compared with the longitudinal direction,
whereas the CH2 intensity does not change substan-
tially. This implies that the OH intensity from
cellulose microfibrils has a strong polarization depen-
dence in outer scales; this trend was not observed for
inner scales. Polarization dependence of second-
harmonic generation ðxout ¼ 2  xin Þ has been
observed for cellulose microfibrils in algal and plant
cell walls (Brown Jr et al. 2003; Cox et al. 2005). In
previous SFG study of flax, the orientation of cellulose
microfibrils was shown to correlate with the SFG
intensity of the OH peak at 3,320 cm-1 (Lee et al.
2013a). When the laser incidence plane with p-polar-
ized IR was aligned along the flax fiber direction (the
direction of most cellulose microfibrils), the
3,320 cm-1 SFG intensity was significantly enhanced Fig. 7 Ratio of peak areas in the CH2 and OH stretch regions of
(Lee et al. 2013a). Similarly, the OH SFG peak the SFG spectra of abaxial cell walls obtained along the
longitudinal and transverse directions of the cell. Error bar is
intensity was substantially enhanced when the p-polar- SEM (n = 10)
ized IR beam was aligned along the cellulose chain
axis within uniaxially aligned cellulose Ib nanowhis-
kers; the orientation dependence of the OH SFG peak cellulose in primary cell walls (Fig. S4 in Electronic
was also consistent with the alignment orientation Supplementary Material).
found by two-dimensional (2D) X-ray diffraction The 3,320 cm-1 peak originates from the hydroxyl
(XRD) analysis of the same samples (Lee et al. groups involved in intrachain hydrogen bonding along
2013b). Similar 2D XRD analysis results for the onion the chain (Lee et al. 2013a, b); thus, the strong
epidermis samples were not as clear as for those model enhancement of this peak upon the alignment of the
systems (Fig. S3 in Electronic Supplementary Mate- input IR polarization along the transverse direction of
rial); this might be due to the low crystallinity of the the cell indicates that cellulose microfibrils in the

123
1084
second abaxial layer of the onion bulb are preferen-
tially aligned along the transverse direction. The
degree of net orientation is the greatest in the most
mature (outer) scales and gradually decreases for
younger (inner) scales (Fig. 7); this trend is consistent
with the progressive changes of surface microfibrils
observed by AFM (Fig. 4).
It is important to note that the SFG signal comes
from the cellulose microfibrils within the entire cell
wall thickness. The observation of polarization depen-
dence of SFG implies that the average orientation of
cellulose microfibrils in the outer epidermal cell walls
is along the transverse direction. Within the cell wall,
the microfibrils near the cuticle side were deposited
earlier and those near the plasma membrane side were
deposited most recently. Although the preferential
alignment of cellulose microfibrils averaged across the
cell wall appears to be similar to the surface orienta-
tion found by AFM imaging, how the microfibril
direction progresses from the membrane side to the
cuticle side cannot be determined from the current
study. The deposition history of microfibril orienta-
tions or their rearrangement should be obtained
independently through measurements of microfibril
orientations of the same scales collected at various
growth stages and mechanical strains.
Conclusions
Here we report the use of AFM imaging and SFG
spectroscopic techniques for the study of cellulose
microfibril orientation in abaxial epidermal walls of
onion scales. SFG spectroscopy can selectively probe
the orientation of cellulose microfibrils without spec-
tral interferences from cell wall matrix polysaccha-
rides in intact plant walls, while AFM reveals the
orientation of microfibrils exposed at the cell wall
surface. For abaxial epidermal walls of onion bulb, the
net orientation of cellulose microfibrils across the
entire wall thickness seems to vary gradually from a
dispersed arrangement in the inner scales to transverse
orientation in the outer scales. A similar trend is
observed for the orientations of cellulose microfibrils
exposed at the newly deposited cell wall surface.
Acknowledgments This work was supported by The Center
for Lignocellulose Structure and Formation, an Energy Frontier
Research Center funded by the US Department of Energy,
123
Cellulose (2014) 21:1075–1086
Office of Science, and Office of Basic Energy Sciences under
award number DE-SC0001090. We acknowledge Anthony J.
Barthel for help with optical profilometry measurements, Liza
Wilson with FTIR, and Lin Fang with 2D XRD measurements.
References
Anderson CT, Carroll A, Akhmetova L, Somerville C (2010)
Real-time imaging of cellulose reorientation during cell
wall expansion in Arabidopsis roots. Plant Physiol
152(2):787–796
Barnette AL, Bradley LC, Veres BD, Schreiner EP, Park YB,
Park J, Park S, Kim SH (2011) Selective detection of
crystalline cellulose in plant cell walls with sum-fre-
quency-generation (SFG) vibration spectroscopy. Bio-
macromolecules 12(7):2434–2439
Barnette AL, Lee C, Bradley LC, Schreiner EP, Park YB, Shin
H, Cosgrove DJ, Park S, Kim SH (2012) Quantification of
crystalline cellulose in lignocellulosic biomass using sum
frequency generation (SFG) vibration spectroscopy and
comparison with other analytical methods. Carbohydr
Polym 89(3):802–809
Baskin T (2005) Anisotropic expansion of the plant cell wall.
Annu Rev Cell Dev Biol 21:203–222
Brown RM Jr, Millard AC, Campagnola PJ (2003) Macromo-
lecular structure of cellulose studied by second-harmonic
generation imaging microscopy. Opt Lett 28(22):2207–2209
Chen L, Wilson RH, McCann MC (1997) Investigation of
macromolecule orientation in dry and hydrated walls of
single onion epidermal cells by FTIR microspectroscopy.
J Mol Struct 408:257–260
Cosgrove DJ (2000) Expansive growth of plant cell walls. Plant
Physiol Biochem 38(1):109–124
Cox G, Moreno N, Feijó J (2005) Second-harmonic imaging of
plant polysaccharides. J Biomed Opt 10(2):024013
Davies LM, Harris PJ (2003) Atomic force microscopy of
microfibrils in primary cell walls. Planta 217(2):283–289
Ding SY, Himmel ME (2006) The maize primary cell wall
microfibril: a new model derived from direct visualization.
J Agric Food Chem 54(3):597–606
Dubois M, Gilles KA, Hamilton JK, Rebers P, Smith F (1956)
Colorimetric method for determination of sugars and
related substances. Anal Chem 28(3):350–356
Fernandes AN, Thomas LH, Altaner CM, Callow P, Forsyth VT,
Apperley DC, Kennedy CJ, Jarvis MC (2011) Nanostruc-
ture of cellulose microfibrils in spruce wood. PNAS
108(47):E1195–E1203
Giddings TH Jr, Staehelin LA (1988) Spatial relationship
between microtubules and plasma-membrane rosettes
during the deposition of primary wall microfibrils in
Closterium sp. Planta 173(1):22–30
Green PB (1960) Multinet growth in the cell wall of Nitella.
J Biophys Biochem Cytol 7(2):289–296
Green PB (1962) Mechanism for plant cellular morphogenesis.
Science 138(3548):1404–1405
Green P (1964) Cell walls and the geometry of plant growth.
Brookhaven Symp Biol 16:203–217
Ha MA, Apperley DC, Jarvis MC (1997) Molecular rigidity in dry
and hydrated onion cell walls. Plant Physiol 115(2):593–598
Cellulose (2014) 21:1075–1086
Heath IB (1974) A unified hypothesis for the role of membrane
bound enzyme complexes and microtubules in plant cell
wall synthesis. J Theor Biol 48(2):445–449
Hieu HC, Tuan NA, Li H, Miyauchi Y, Mizutani G (2011) Sum
frequency generation microscopy study of cellulose fibers.
Appl Spectrosc 65(11):1254–1259
Hutter JL, Bechhoefer J (1993) Calibration of atomic-force
microscope tips. Rev Sci Instrum 64:1868
Kutschera U (2008) The growing outer epidermal wall: design
and physiological role of a composite structure. Ann Bot
101(5):615–621
LaComb R, Nadiarnykh O, Townsend SS, Campagnola PJ
(2008) Phase matching considerations in second harmonic
generation from tissues: effects on emission directionality,
conversion efficiency and observed morphology. Opt
Commun 281(7):1823–1832
Lambert AG, Davies PB, Neivandt DJ (2005) Implementing the
theory of sum frequency generation vibrational spectros-
copy: a tutorial review. Appl Spectrosc Rev 40(2):103–145
Lee CM, Mittal A, Barnette AL, Kafle K, Park Y, Shin H, Johnson
DK, Park S, Kim SH (2013a) Cellulose polymorphism study
with sum-frequency-generation (SFG) vibration spectros-
copy: identification of exocyclic CH2OH conformation and
chain orientation. Cellulose 20(3):991–1000
Lee CM, Mohamed NMA, Watts HD, Kubicki JD, Kim SH
(2013b) Sum-frequency-generation vibration spectroscopy
and density functional theory calculations with dispersion
corrections (DFT-D2) for cellulose Ia and Ib. J Phys Chem
B 117(22):6681–6692
Li S, Gu Y (2012) Cellulose biosynthesis in higher plants and
the role of the cytoskeleton. In: Hetherington AM (ed) eLS.
Wiley, Chichester, pp 1–8
Marechal Y, Chanzy H (2000) The hydrogen bond network in Ib
cellulose as observed by infrared spectrometry. J Mol
Struct 523(1):183–196
Marga F, Grandbois M, Cosgrove DJ, Baskin TI (2005) Cell wall
extension results in the coordinate separation of parallel
microfibrils: evidence from scanning electron microscopy
and atomic force microscopy. Plant J 43(2):181–190
McCann M, Wells B, Roberts K (1990) Direct visualization of
cross-links in the primary plant cell wall. J Cell Sci
96(2):323–334
Mita T, Shibaoka H (1983) Changes in microtubules in onion
leaf sheath cells during bulb development. Plant Cell
Physiol 24(1):109–117
Neville A (1985) Molecular and mechanical aspects of helicoid
development in plant cell walls. BioEssays 3(1):4–8
Neville A, Gubb D, Crawford R (1976) A new model for cel-
lulose architecture in some plant cell walls. Protoplasma
90(3–4):307–317
Ng A, Parker ML, Parr AJ, Saunders PK, Smith AC, Waldron KW
(2000) Physicochemical characteristics of onion (Allium
cepa L.) tissues. J Agric Food Chem 48(11):5612–5617
Paredez AR, Somerville CR, Ehrhardt DW (2006) Visualization
of cellulose synthase demonstrates functional association
with microtubules. Science 312(5779):1491–1495
Park YB, Lee CM, Koo B-W, Park S, Cosgrove DJ, Kim SH
(2013) Monitoring meso-scale ordering of cellulose in
intact plant cell walls using sum frequency generation
spectroscopy. Plant Physiol 163(2):907–913
1085
Preston RD (1974) The physical biology of plant cell walls.
Chapman & Hall, London
Richmond PA, Métraux JP, Taiz L (1980) Cell expansion pat-
terns and directionality of wall mechanical properties in
Nitella. Plant Physiol 65(2):211–217
Roelofsen PA, Houwink A (1953) Architecture and growth of
the primary cell wall in some plant hairs and in the Phyc-
omyces sporangiophore. Acta Bot Ner 2:218–225
Roland J-C, Vian B, Reis D (1977) Further observations on cell
wall morphogenesis and polysaccharide arrangement dur-
ing plant growth. Protoplasma 91(2):125–141
Satiat-Jeunemaifre B, Martin B, Hawes C (1992) Plant cell wall
architecture is revealed by rapid-freezing and deep-etch-
ing. Protoplasma 167(1–2):33–42
Sugimoto K, Williamson RE, Wasteneys GO (2000) New
techniques enable comparative analysis of microtubule
orientation, wall texture, and growth rate in intact roots of
Arabidopsis. Plant Physiol 124(4):1493–1506
Suslov D, Verbelen JP, Vissenberg K (2009) Onion epidermis as
a new model to study the control of growth anisotropy in
higher plants. J Exp Bot 60(14):4175–4187
Thimm JC, Burritt DJ, Ducker WA, Melton LD (2000) Celery
(Apium graveolens L.) parenchyma cell walls examined by
atomic force microscopy: effect of dehydration on cellu-
lose microfibrils. Planta 212(1):25–32
Thomas LH, Forsyth VT, Šturcová A, Kennedy CJ, May RP,
Altaner CM, Apperley DC, Wess TJ, Jarvis MC (2013)
Structure of cellulose microfibrils in primary cell walls
from collenchyma. Plant Physiol 161(1):465–476
Wang H-F, Gan W, Lu R, Rao Y, Wu B-H (2005) Quantitative
spectral and orientational analysis in surface sum fre-
quency generation vibrational spectroscopy (SFG-VS). Int
Rev Phys Chem 24(2):191–256
Wellner N, Kačuráková M, Malovı́ková A, Wilson RH,
Belton PS (1998) FT-IR study of pectate and pectinate gels
formed by divalent cations. Carbohydr Res 308(1):
123–131
Wells B, McCann M, Shedletzky E, Delmer D, Roberts K
(1994) Structural features of cell walls from tomato cells
adapted to grow on the herbicide 2,6-dichlorobenzonitrile.
J Microsc 173(2):155–164
Wiley JH, Atalla RH (1987) Band assignments in the Raman
spectra of celluloses. Carbohydr Res 160:113–129
Wilson RH, Smith AC, Kačuráková M, Saunders PK, Wellner
N, Waldron KW (2000) The mechanical properties and
molecular dynamics of plant cell wall polysaccharides
studied by Fourier-transform infrared spectroscopy. Plant
Physiol 124(1):397–406
Wood PJ (1980) Specificity in the interaction of direct dyes with
polysaccharides. Carbohydr Res 85(2):271–287
Wood PJ, Fulcher R, Stone BA (1983) Studies on the specifi-
city of interaction of cereal cell wall components with
Congo Red and Calcofluor. Specific detection and histo-
chemistry of (1 ? 3), (1 ? 4),-b-D-glucan. J Cereal Sci
1(2):95–110
Yoneda A, Ito T, Higaki T, Kutsuna N, Saito T, Ishimizu T,
Osada H, Hasezawa S, Matsui M, Demura T (2010) Cob-
torin target analysis reveals that pectin functions in the
deposition of cellulose microfibrils in parallel with cortical
microtubules. Plant J 64(4):657–667
123
1086 Cellulose (2014) 21:1075–1086

Zhang T, Mahgsoudy-Louyeh S, Tittmann B, Cosgrove D
(2013) Visualization of the nanoscale pattern of recently-
deposited cellulose microfibrils and matrix materials in
never-dried primary walls of the onion epidermis. Cellu-
lose 1–10. doi:10.1007/s10570-013-9996-1
Zugenmaier P (2008) Crystalline cellulose and derivatives. In:
Timell TE, Wimmer R (eds) Springer series in wood sci-
ence. Springer, Berlin, pp 101–174

123