Journal of Experimental Botany, Vol. 55, No. 397, pp. 711±718, March 2004
DOI: 10.1093/jxb/erh077 Advance Access publication 13 February, 2004
RESEARCH PAPER
Self assembly of epicuticular waxes on living plant
surfaces imaged by atomic force microscopy (AFM)
Kerstin Koch1,*, Christoph Neinhuis2, Hans-JuÈrgen Ensikat1 and Wilhelm Barthlott1
1
Nees-Institut fuÈr BiodiversitaÈt der P¯anzen, UniversitaÈt Bonn, Meckenheimer Allee 170, D-53115 Bonn,
Germany
2
Institut fuÈr Botanik, Technische UniversitaÈt Dresden, Zellescher Weg 22, D-01062 Dresden, Germany
Received 3 September 2003; Accepted 2 December 2003
Abstract
The cuticle of terrestrial vascular plants and some
bryophytes is covered with a complex mixture
of lipids, usually called epicuticular waxes. Selfassembly processes of wax molecules lead to
crystalline three-dimensional micro- and nanostructures that emerge from an underlying wax ®lm. This
paper presents the ®rst AFM study on wax regeneration on the surfaces of living plants and the very
early stages of wax crystal formation at the molecular
level. Wax formation was analysed on the leaves of
Euphorbia lathyris, Galanthus nivalis, and Ipheion
uni¯orum. Immediately after wax removal, regeneration of a wax ®lm began, consisting of individual
layers of, typically, 3±5 nm thickness. Subsequently,
several different stages of crystal growth could be
distinguished, and different patterns of wax regeneration as well as considerable variation in regeneration speed were found.
Key words: Atomic force microscopy (AFM), crystallization,
epicuticular waxes, nanostructures, regeneration, scanning
electron microscopy (SEM), self assembly.
Introduction
The cuticle of terrestrial vascular plants and some
bryophytes consists mainly of two hydrophobic
components, the biopolymer cutin and a mixture of lipids
(Martin and Juniper, 1970; Jeffree, 1996; Kolattukudy,
1980, 2001; Bargel et al., 2003), which are usually called
`waxes'. Waxes are embedded in the polymer matrix
(intracuticular) and also deposited upon the surface
(epicuticular waxes). Epicuticular waxes form the outermost boundary layer of the plant, representing a multifunctional interface between plant and environment. A
major function is to serve as a barrier against uncontrolled
water loss (SchoÈnherr, 1976, 1982; Riederer and Schreiber,
1995). Three-dimensional wax crystals constitute a
hydrophobic micro-structured surface layer. Due to their
chemistry and microstructure, wax crystals form a
hydrophobic water-repellent surface. Such surfaces often
display a self-cleaning property, called the `lotus-effect',
by increased water repellency and reduced adhesion of the
contaminating particles (Barthlott and Neinhuis, 1997). In
some cases waxes cause an increase in the re¯ection of
solar radiation (Barnes and Cardoso-Vilhena, 1996;
Holmes and Keiller, 2002).
The ®rst person to describe waxes on plant surfaces,
using light microscopy, and term them `kristalloids'
(crystalloids) was De Bary (1871). Starting with Juniper
and Bradley (1958), Amelunxen et al. (1967), Jeffree et al.
(1975, 1976), and Baker (1982), epicuticular waxes have
been investigated intensely. More recently, the crystalline
nature of the wax of many species has been veri®ed by Xray powder diffractograms and electron diffraction
(Reynhardt, 1997; Reynhardt and Riederer, 1988, 1994;
Meusel et al., 1994, 1999, 2000). In general, most plant
waxes are a complex mixture of long-chain aliphatic
components, for example, primary and secondary alcohols,
aldehydes, and ketones, while others are dominated by
cyclic components like triterpenes or ¯avonoids (overviews in Barthlott and Wollenweber, 1981; Baker, 1982;
Jeffree, 1986; Barthlott, 1990; Walton, 1990). Epicuticular
waxes form thin ®lms or thick crusts and often superimposed three-dimensional structures on an underlying wax
®lm (Kolattukudy, 1980; Bianchi, 1995; Barthlott et al.,
* To whom correspondence should be addressed. Fax: +49 (0)228 733120. E-mail: [email protected]
Abbreviations: AFM, atomic force microscopy; SEM, scanning electron microscopy.
Journal of Experimental Botany, Vol. 55, No. 397, ã Society for Experimental Biology 2004; all rights reserved
712 Koch et al.
1998, 2003; Barthlott, 1990). Because waxes are crystalline, the varying shape of the crystals is determined by
their chemical composition. In some types of wax crystal,
the ultrastructure is determined by one predominating wax
component (Jeffree et al., 1975, 1976). A well-known
example are the nonacosan-10-ol dominated tubules (Jetter
and Riederer, 1994, 1995). On the other hand, there are
wax crystals that are determined by a minor component,
such as those described for longitudinal ridged rodlets
(Meusel et al. 2000). Nevertheless, due to their small size,
the chemical composition of an individual wax crystal is
still hypothetical. AFM nowadays facilitates the observation of dynamic crystallization processes of organic and
inorganic molecules under real environmental conditions
(Becker and Gasharova, 2001). The ability of AFM to
image a wide variety of samples under variable conditions,
has created a great interest in applying it to the study of
biological structures (Hoh and Hansma, 1992; Morris et al.,
1999).
Materials and methods
Selection of plant taxa
For long-term investigations of wax regeneration by AFM the size
and shape of the leaves must allow the montage in the AFM without
cutting the leaves. Optimal leaf sizes and surface evenness were
found on leaves of Euphorbia lathyris L. (Euphorbiacea), Galanthus
nivalis L. (Amaryllidaceae), and Ipheion uni¯orum Grah.
(Liliaceae). These plants were cultivated in the Botanical Gardens,
University of Bonn. For the examinations, the plants were potted and
transferred to the laboratory.
Wax regeneration
Wax regeneration was investigated on adaxial leaf surfaces after
removing the original wax layer. To obtain a completely clean
surface, the areas (c. 3 mm diameter) were dewaxed twice by
applying a drop of two component glue (UHU-plus-Schnellfest 2Komponenten Epoxidharz-Kleber, Henkel DuÈsseldorf, Germany)
onto the leaf and then the removal of the hardened ®lm. As very
young leaves which are just unfolding from the bud are often
damaged during dewaxing, young expanding leaves with a length of
4±5 cm were investigated. Galanthus and Ipheion were chosen for
long-term observation (6 d) of wax regeneration by SEM. Therefore,
20 leaves of each species were partially dewaxed as described above.
Directly after wax removal the investigation of wax regeneration
began. Wax regeneration was documented every hour during the
subsequent period, which lasted up to 6 h. After 6 h the interval
was increased and then regeneration was monitored every 24 h for up
to 6 d.
Wax extraction for in vitro measurements
Wax from Galanthus leaves was isolated by dipping the leaves into
chloroform (Merck, p.a., Darmstadt) for 30 s. A piece of a silicon
wafer was dipped into a 0.2% solution of wax and chloroform. After
evaporation of the solvent the wax deposit was investigated by AFM.
Atomic force microscopy (AFM)
A Nano Scope III (Digital Instruments, Mannheim, Germany) with a
z-piezo with 10 mm range was used. Studies were made in the
tapping mode with silicon tapping-mode-tips. To prevent thermal
effects of the AFM-laser-beam on the plant surface, cantilevers with
re¯ective coating were used. The beam intensity was reduced by
integrating an attenuation ®lter above the cantilever. The plants were
placed near the AFM so that the apical part of the leaf could be
mounted on a sample holder plate with the same glue as used for wax
removal, placing the dewaxed area in the centre. After the second
dewaxing procedure, the leaf was immediately placed in the AFM,
where the ®rst image could be obtained after a few minutes. Wax
layers are usually relatively soft, and they can be rapidly damaged at
higher magni®cations, employing a scan size of less than 1 mm.
Appropriate AFM-conditions turned out to be a scan size of 3±20 mm,
a scan rate of 0.7±2 lines s±1 encompassing 256 lines per image, and
a setpoint near the upper limit to minimize the interaction between
tip and sample. Measurements of height and length of the waxes
were made with the integrated AFM tool `Section Analysis'.
Scanning electron microscopy (SEM)
A LEO 440i microscope (Leica, Bensheim, Germany) was used for
SEM. Fresh leaves were mounted on aluminium stubs with doublesided adhesive tape and coated with gold (25 nm) in a sputter coater
(SCD-040; Balzers, Wiesbaden, Germany), followed by SEM
examinations at 15 kV.
Results
The study of the growth of wax layers and crystals in situ
showed that regeneration starts immediately after removing the wax. Considerable differences were found in the
speed of regeneration and in the development of the wax
®lms.
Euphorbia lathyris
The ®rst AFM image was obtained 5 min after removal
of the wax. In this early stage patches of wax layers of
c. 0.3 mm in diameter were present. Further wax patches
appeared at different spots on the surface (Fig. 1a). From
these initial wax patches the layers started to expand. The
selected pictures in Fig. 1 show that patches expanded to
irregular islands, and demonstrate the lateral accumulation
of new wax. Within about 90 min. the wax layer covered
nearly 15% of the total area (9 mm2). These layers grew
continuously, so that after about 3 h c. 30% of the cuticle
was covered with wax. Approximately 10 h after removal,
a complete wax ®lm was regenerated, and after 20 h a
multilayered wax ®lm could be detected (Fig. 1d). The
thickness of the growing layer was between 3.4 nm and
7 nm, but measurements with the `Section Analysis'
software were inaccurate due to the roughness of the leaf
surface. The estimated real thickness must be between 4
and 5 nm. However, no wax crystals comparable to those
seen on the intact leaf surface were regenerated within the
time mentioned.
Galanthus nivalis
AFM examination of the wax ®lm regeneration: Flat wax
patches were observed immediately after the ®rst scan was
®nished, 4 min 30 s after removing the wax. The waxes
initially appeared at several spots on the cuticle. This layer
grew laterally at certain spots, thus irregular patterns of
Self assembly of epicuticular waxes 713
Fig. 1. (a±d) Euphorbia lathyris selected AFM images (area 333 mm) of wax regeneration. (a) After 1 h 38 min, the regenerated waxes are
coloured in red. (b) After 2 h 11 min, further accumulated wax is coloured in yellow. (c) After 3 h 3 min, additional accumulated wax is coloured
in green. (d) 20 h after wax removal a multilayered wax ®lm was regenerated.
branched stripes arose (Fig. 3). Before the ®rst wax layer
completely covered the cuticle, the second wax layer
started to grow (Fig. 3b). The pattern of the second wax
layer was similar to the ®rst one. After 80 min the cuticle
was completely covered with a wax ®lm, composed of two
distinct layers (Fig. 3d). The thickness of each single layer
(height of steps) seems to be similar, but the thickness
measurement was inaccurate for the same reasons as in
Euphorbia. Measured values varied between 5 nm and
11 nm, but the estimated real thickness must be between
8 nm and 10 nm.
To prove the reliability of these measurements, in vitro
crystallized waxes from Galanthus were measured on a
smooth silicon surface (Fig. 2). The thickness of the in vitro
crystallized wax layers ranged from 3.7±4.5 nm, with an
average value of 4.3 nm (n=18). Some layers varied
between 8.1 and 9.1 nm, with an average value of 8.72 nm
(n=16).
AFM study of crystal growth: The ®rst image of growing
crystals was taken 13 min after dewaxing. At this time, the
biggest crystal reached a maximal height of 77.6 nm, and
approximately 20% of the dewaxed surface area was
covered with a wax ®lm (Fig. 3a). The increase in crystal
size was measured for the rapidly growing crystal (Fig. 3c,
d). Within a period of 67 min, a length increase from
1.90 mm to 4.46 mm was measured. Different stages of
growth are illustrated in Fig. 4.
SEM study of wax regeneration: Immediately after
removal of the wax, leaves were also examined by SEM.
In these early stages no regenerated waxes or residues of
714 Koch et al.
Fig. 2. Height measurment of the in vitro crystallized waxes from Galanthus, on a silicon surface. (Left) AFM height picture with the integrated
line of measured points. (Right) Pro®le view of the line section. The vertical distance of the insert markers was 4.28 nm and represents the height
of a step in the wax.
Fig. 3. (a±d) Galanthus nivalis selected AFM images (636 mm area) representing the wax regeneration within a time scale of 80 min. Arrows
show signi®cant changes of wax formation; the white arrow is an orientation point to demonstrate the length increase of the wax crystal. The
black arrow marks a wax crystal, which was later removed by the AFM tip. For better detection, different wax layers are coloured individually;
the grey areas are cuticle surface, the ®rst wax layer is shown in brown, and the second layer in green. The higher crystals are marked in violet.
wax were detectable compared with observing the early
stages in the AFM (Fig. 5b). However, because of the lack
of the height resolution the wax ®lms were not visible by
SEM. The ®rst regenerated wax was visible after 4 h. After
24 h the surface was evenly covered with granule like
waxes (Fig. 5c). Observation was then continued for 6 d.
Within this time nearly 40% of the initial amount of wax
could be observed (Fig. 5d) but the regenerated wax
Self assembly of epicuticular waxes 715
Fig. 4. Outlines of the wax crystal that was marked by a white arrow in Fig. 3d. The accumulation of new wax material during the crystal growth
is demonstrated by the overlay of the outlines at different stages.
crystals were smaller and formed ®lamentous platelets
instead of the `longitudinal ridged rodlets' in the intact leaf
area (Fig. 5a).
Ipheion uni¯orum
In this species the AFM examination showed a more
complex growth process during wax regeneration.
Immediately after removal, the surface still seemed to be
covered by ¯at, irregularly formed wax stripes. These
stripes resembled those observed on Galanthus leaves
(Fig. 3a, b). The observation of a layer different in size and
growth from the layers observed on Ipheion and
Galanthus, was remarkable. This layer spread rapidly
over the surface and covered even higher surface sculptures (encircled in Fig. 6a, b), but did not overlap the
already existing stripes. The layer varied in thickness from
2 nm to 12 nm. Within these experiments rapidly growing
crystals were observed. Sometimes single crystals were
pushed away by the AFM tip, but in the latter a new crystal
emerged at the same place after a few minutes. Like in the
other species, wax regeneration was studied by SEM. After
6 d nearly 70% of all formerly existing wax crystals were
regenerated as platelets, similar to the original leaf surface.
Discussion
Considering the vital role of waxes as a protective,
hydrophobic boundary layer (Riederer and Schreiber,
1995; Kirkwood, 1999), regeneration of a removed or
damaged wax ®lm is obviously of great importance. This
assumption was proved by the data obtained from all three
species, as they all started to regenerate a wax ®lm of
several layers immediately after removal of the wax.
Because plant cuticles are not completely smooth, it was
dif®cult to measure the thickness of the wax ®lm exactly.
Thus, the given values for wax layer thickness on leaves
are rather rough estimations. Only on very smooth
substrates like polished silicon wafers, can the thickness
of deposited wax layers be measured exactly. On silicon
substrates, the thickness of the layers was found to be
typical for the individual kind of crystallized wax, which
could be veri®ed with in vitro crystallized wax from the
same species. From X-ray diffraction studies it is known
that some plant epicuticular waxes crystallize in a
bilayered structure, while others form a single-layer
structure ((Reynhardt, 1997). Although the measurements
on the leaf surfaces were coarse, it was assumed that the
®rst wax layers on Galanthus leaves were bilayers and on
Euphorbia leaves were monolayers, while the chain
lengths of the molecules from 20±40 carbon atoms
correspond to a length of c. 2.4±4.8 nm.
Galanthus wax crystallized in vitro on a silicon substrate
shows both single and double layers. The measured layer
thickness, with an average value of 4.3 nm, is in
accordance with the chain length of the most common
aliphatic wax components (C28±C34) and may indicate a
monolayer. Nevertheless, it could be that these layers are
double layers of short aliphatic wax compounds, as C16
and C18 fatty acids or primary alcohols. In addition to the
wax layers, the growth of three-dimensional crystals was
observed. These taller wax sculptures grew directly on the
cuticle or on the wax ®lm. It may therefore be concluded
that the independent growth processes between the wax
®lm and the wax crystals is a separation of different
chemical compounds. In vitro crystallization experiments
with isolated wax compounds have shown that sometimes
only one compound of the wax mixture is critical for the
formation of a certain wax type. Different authors (Jeffree
et al., 1975, 1976; Jetter and Riederer, 1995) demonstrated
that nonacosanol is the compound within the wax mixture
responsible for tubule formation. In addition, a separation
of wax components with respect to chain length and
polarity of the molecules has also been detected by X-ray
diffraction (Reynhardt, 1997). High resolution AFM
716 Koch et al.
Fig. 5. (a±d) SEM pictures from the upper side of Galanthus leaves. (a) Longitudinal ridged rodlets on the intact leaf. (b) Leaf surface 10 min
after removing the wax. (c) 24 h after wax removal, granule waxes were regenerated. (d) 6 d after wax removal, ®lamentous waxes were
regenerated.
Fig. 6. (a, b) Ipheion uni¯orum selected AFM images (535 mm area) of wax regeneration. The pictures represent two stages of growing wax
stripes and a rapidly growing layer at 40 min and 49 min after removal of the epicuticular wax.
Self assembly of epicuticular waxes 717
measurements of in vitro-grown isolated wax components
are necessary to establish whether the formation of threedimensional wax crystals on the wax ®lm is also a phase
separation process.
The observed considerable differences in the speed and
intensity of wax regeneration could be caused by different
reasons. For several species it has previously been shown
that young leaves regenerate epicuticular waxes particularly well (Hallam, 1967, 1970; Wolter et al., 1988; Percy
and Baker, 1987; Wirthensohn and Sedgley, 1996;
Neinhuis et al., 2001; Jetter and SchaÈfer, 2001). For this
reason, in this study only young, expanding leaves were
used. However, on leaves of Euphorbia the regeneration of
the wax ®lm was nearly 12 times slower than that on
Galanthus leaves. Several authors discussed the hypothetical transport mechanism of wax through the cuticle (PostBeitenmiller, 1996). Recently, a co-transport mechanism
of wax and water was discussed by Neinhuis et al. (2001).
There, the driving force for the movement of the wax
molecules could be the higher transpiration rate in the
dewaxed area. The high amount of regenerated waxes after
6 d indicates that new wax was synthesized, but the
regeneration of the ®rst wax layers of the wax ®lm could be
the result of diffusion of intracuticular wax onto the
cuticle.
As well as the amount of available wax, the mobility of
wax molecules on their way through and onto the cuticle
could in¯uence the speed of regeneration. AFM examination of the crystals on Galanthus leaves have shown that
in the early phase crystals have grown preferentially
horizontally and in one main direction. AFM analyses have
shown that wax material moves to certain spots to the
cuticle, and thus lateral movement of the wax molecules to
the edges and steps of the growing layers and crystals
seems to be essential. In addition, in Ipheion and
Galanthus, the growth of crystals on a closed wax layer
was observed, which indicates that further wax molecules
must cross this layer. Reynhardt and Riederer (1994)
emphasized the importance of crystalline and amorphous
zones in plant waxes in regulating the diffusion of
molecules across the transport barrier. (Reynhardt (1997)
demonstrated that a mixture of short and long chains form
amorphous zones of higher ¯uidity. These amorphous
zones could be the pathways for the new wax molecules on
their way through a wax ®lm. However, for the growth of
multilayered wax ®lms, it is not clear whether the second
and the following layers grow over or under the ®rst one.
Growth by insertion of material beneath the upper layer
may seem unusual, but must be considered since the supply
of wax molecules comes from inside the cuticle. The
simultaneous growth of a layer over the Ipheion leaf,
apparently without altering or obscuring the pre-existing
and still growing wax stripes, may indicate that the growth
of layers does not just occur by the addition of material to
the surface. The low contrast in the AFM-image of some
edges of wax layers suggests that they are located below
the super®cial layer. When crystals started to grow
vertically, they were often damaged by the AFM probe.
Thus, SEM is the more reliable tool to detect the ®nal
shape of the regenerated wax crystals.
Conclusion
From the AFM examinations presented here, it is concluded that regeneration of epicuticular lipids on living
plant surfaces is a highly dynamic and comparatively swift
process, re¯ecting the importance of a steadily present
continuous outer leaf coverage. Although AFM studies are
limited by surface roughness and the fragility of soft
structures like epicuticular waxes, the AFM provides new
opportunities to detect and image minimal amounts of wax
such as in the form of thin monomolecular layers, not
detectable by SEM.
Acknowledgements
The authors would like to thank the Deutsche Forschungsgemeinschaft (DFG), Deutsche Bundesstiftung Umwelt (DBU), and the
Bundesministerium fuÈr Bildung und Forschung (BMBF) for
®nancial support. We would also like to thank H Bargel (TU
Dresden) for his critical comments and discussions.
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