Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
Phil. Trans. R. Soc. A (2009) 367, 1673–1688
doi:10.1098/rsta.2009.0015
Self-healing of voids in the wax coating on
plant surfaces
B Y K ERSTIN K OCH 1, * , B HARAT B HUSHAN 2 , H ANS -J ÜRGEN E NSIKAT 1
1
AND W ILHELM B ARTHLOTT
1
Nees Institute for Biodiversity of Plants, Rheinische Friedrich-Wilhelms
University of Bonn, Meckenheimer Allee 170, 53115 Bonn, Germany
2
Nanoprobe Laboratory for Bio- & Nanotechnology and Biomimetics,
Ohio State University, 201 West 19th Avenue, Columbus,
OH 43210, USA
The cuticles of plants provide a multifunctional interface between the plants and their
environments. The cuticle, with its associated waxes, is a protective layer that minimizes
water loss by transpiration and provides several functions, such as hydrophobicity, light
reflection and absorption of harmful radiation. The self-healing of voids in the
epicuticular wax layer has been studied in 17 living plants by atomic force microscopy
(AFM), and the process of wax film formation is described. Two modes of wax film
formation, a concentric layer formation and striped layer formation, were found, and the
process of multilayer wax film formation is discussed. A new method for the preparation
of small pieces of fresh, water-containing plant specimens for AFM investigations is
introduced. The technique allows AFM investigations of several hours duration without
significant shrinkage or lateral drift of the specimen. This research shows how plants
refill voids in their surface wax layers by wax self-assembly and should be useful for the
design of self-healing materials.
Keywords: self-healing; self-assembly; epicuticular waxes; atomic force microscopy;
specimen preparation
1. Introduction
The epidermis, as the outermost cell layer of the primary tissues of all leaves and
several other organs of plants, plays an important role in environmental interactions
and surface structuring. The outer part of epidermis cells, schematically shown in
figure 1, is an extracellular membrane called the cuticle. The cuticle is basically a
biopolymer, composed of cutin and integrated and superimposed waxes. Waxes
form the main transport barrier to reduce the loss of water by transpiration
and reduce leaching of molecules from inside the cells (Riederer & Schreiber 1995,
2001). Wax on the cuticle is called epicuticular wax; wax located in the cutin
network is called intracuticular wax. Epicuticular waxes play an important role
in surface wettability (Barthlott & Neinhuis 1997; Koch et al. 2008). In some
* Author for correspondence ( [email protected]).
One contribution of 9 to a Theme Issue ‘Biomimetics II: fabrication and applications’.
1673
This journal is q 2009 The Royal Society
Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
1674
K. Koch et al.
(a)
50 µm
(b)
epicuticular wax
cuticula with intracuticular wax
pectin
cell wall
plasma membrane
Figure 1. (a) SEM of a leaf surface of Euphorbia characias after partial (right part of the figure)
removal of the epicuticular waxes. (b) The schematic shows the stratification of the outermost
layers of the plant epidermis cells. The epicuticular wax layer is shown as a composite of threedimensional waxes with an underlying wax film. The cuticle with its integrated waxes is connected
with the underlying cellulose wall by pectin, here simply visualized as a layer. Below the
cell wall, the plasma membrane is shown. It separates the water-containing part of the epidermis
cell from the outermost components of the epidermis above. It separates the living cytoplasm from
the cell wall.
species, waxes reduce the adhesion of insects (Gorb et al. 2005) and, in others, they
are responsible for the reflection of visible light and absorption of harmful UV
radiation (Barnes & Cardoso-Vilhena 1996; Koch et al. 2009).
Most plant waxes are a complex mixture of long-chain aliphatic components,
e.g. primary and secondary alcohols, aldehydes and ketones, while others contain
high amounts of cyclic components, such as triterpenes or flavonoids (Jeffree
2006). Epicuticular waxes occur in different morphologies, such as tubules,
platelets, threads and thin films or thicker crusts, which originate by selfassembly (Barthlott et al. 1998). The different morphologies are based on
differences in chemical composition. The combination of three-dimensional wax
crystals with an underlying wax film has been reported for many species (Jeffree
et al. 1975; Barthlott et al. 1998; Koch & Ensikat 2008). The crystalline nature of
the wax of many species has been verified by X-ray and electron diffraction
(Ensikat et al. 2006). Wax films are often incorrectly referred to as an
‘amorphous’ layer, more a morphological description than a crystallographic one.
On several plant surfaces, the wax film consists of only a few molecular layers,
which is hardly visible in the scanning electron microscope (SEM). However, by
mechanical isolation of the epicuticular waxes, e.g. freezing in glycerol (Ensikat
et al. 2000), the waxes can be removed from the cuticle and transferred onto a
smooth artificial substrate for microscopic investigations. By this method, the
edges of the wax film can be detected, and the film thicknesses can be determined
(Koch & Ensikat 2008).
Phil. Trans. R. Soc. A (2009)
Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
Self-healing of voids on plant surfaces
1675
Plant waxes are synthesized inside the living part of the epidermis cells, but
the transport mechanisms of the wax molecules through the outermost
hydrophilic cellulose wall and the cuticle are still under discussion. According
to one hypothesis, epicuticular wax protrusions come out of pores that are
continuous through the cuticle (Baker 1982; Anton et al. 1994), but such pores
were later described as artefacts. More recently, it has been shown that lipidic
compounds, such as waxes, diffuse through the cuticle via a lipidic pathway
(Riederer & Schreiber 1995; Buchholz & Schönherr 2000), whereas water and
polar molecules pass through the cuticle via a polar pathway called ‘aqueous
pores’. Modelling and calculation of the molecular structure of the cuticular
matrix revealed an average aqueous pore radius between 0.3 and 0.5 nm (Popp
et al. 2005; Schönherr 2006). These values are much smaller than the accessible
resolution of atomic force microscopy (AFM) on plant cuticles, and thus such
pores have not been visualized until now. After the wax has moved through
the plant cuticle, the different wax morphologies grow by crystallization or
‘self-assembly’ (Koch et al. 2004; Jeffree 2006). Most epicuticular waxes are
soft and fragile structures and can easily be damaged. Thus, it is of interest to
know whether plants are able to repair voids within the epicuticular wax layer
and whether this process is based on wax self-assembly or other processes.
Epicuticular wax structures usually occur in a range from 0.2 to 100 mm in
thickness; thus, the most suitable microscopy technique for studying the selfassembly process of waxes under ambient conditions is AFM (Koch et al. 2004,
2006a,b). During AFM investigation, loss of water from inside the plant material
should be minimized to reduce specimen drift by cell shrinkage. Water loss can
be reduced by investigation of intact leaves, as shown in figure 2. However, this
precondition limits the selection of species, because most leaves and other organs
of plants are much too large to mount in most AFM specimen chambers without
cutting them.
In this paper, we introduce a new preparation method for AFM investigation of
water-containing leaves and shoots, which allows the cutting of leaves and prevents
desiccation of the specimen for several hours. Previous publications were limited to
observations of a few plant species, which had been examined as entire intact plants
(Koch et al. 2004). Here, we present the results of wax film formation on 17 species.
Wax regeneration was studied in 10 species with intact leaves, and seven species
have been investigated by using small excised pieces of leaves or shoots.
2. Material and methods
(a ) Plant material
All 17 species investigated were cultivated in and provided by the Botanical
Gardens of the Rheinische Friedrich-Wilhelms University of Bonn (BG Bonn).
In table 1, species names, their accession numbers and wax morphologies of the
upper leaf sides (adaxial) are provided.
(b ) Atomic force microscopy
A NanoScope IIIa (Digital Instruments, Mannheim, Germany) with a z-piezo
with 10 mm range was used. Tapping mode and silicon tapping-mode tips
Phil. Trans. R. Soc. A (2009)
Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
1676
K. Koch et al.
Figure 2. Experimental set-up of the AFM for long-term investigations of wax regeneration on a
living plant surface. The tip of the leaf of G. nivalis has been fixed on the specimen holder with a
drop of two-component glue. Existing waxes have been removed and the regeneration (self-healing)
of the wax can be studied over several hours.
(Tap300Al) with a resonant frequency of approximately 300 kHz and force
constant of 40 N mK1 were used (NanoWorld NCHR, JPK Instruments, Berlin,
Germany). The tip radius was approximately 10 nm. To prevent thermal effects
of the AFM laser beam on the plant surface, cantilevers with a reflective
coating were used, and beam intensity was further reduced by integrating an
attenuation filter above the cantilever. Plant waxes are usually relatively fragile
and can be rapidly damaged when scanned at high magnifications, employing a
scan size of less than 1 mm. Appropriate AFM conditions turned out to be a scan
size of 3!3 to 20!20 mm2, a scan rate of 0.7–2 Hz (lines per second) and a
setpoint near the upper limit to minimize the interaction between the tip and
the sample.
(c ) Image processing
The images presented here are ‘amplitude images’, which clearly show the
finer details. For wax film thickness measurements, height images have to be
used, which are recorded simultaneously. The accumulation of waxes has been
visualized by calculating the difference in wax growth between two 16-bit
greyscale images, recorded at different stages during wax regeneration.
Phil. Trans. R. Soc. A (2009)
Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
1677
Self-healing of voids on plant surfaces
Table 1. Plant species, their original wax morphologies of the upper leaf sides (adaxial) and on the
stem of Aporocactus flagelliformis, the observed wax regeneration and the plant accession numbers
from the BG Bonn (annual plant species are temporary in culture). (The type of wax layer
formation during regeneration is indicated as follows: C, concentric layer growth; S, striped layer
growth. The asterisk indicates those species from which only pieces of the leaves or shoots were
used for investigations.)
species (author)
wax type
wax regeneration
Abies alba (Mill.)
film
Aporocactus flagelliformis (Lem.) crusts
Aquilegia canadensis (L.)
film and nonacosanol
tubules
Citrus limon (L.)
film
Coffea arabica (L.)
film
Euphorbia characias (L.)
film and platelets
Euphorbia lathyris (L.)
film and platelets
S-layer formation
C-layer formation
S-layer formation and
nonacosanol tubules
C-layer formation
C-layer formation
C-layer formation
C-layer formation
Galanthus nivalis (L.)
film and platelets
film and platelets
S-layer formation and
rodlets
C-layer formation and
platelets
S-layer formation and
C-layer formation
S-layer formation
Iris germanica (L.)
Ipheion uniflorum (Raf.)
film and rodlets
crusts
Kalanchoe daigremontianum
(Adans.)
Lathyrus odoratus (L.)
Lithops turbiniformis (N.E. Br.)
Leymus arenarius (Hochst.)
Prunus laurocerasus
Taxus baccata
Thalictrum flavum
glaucum (L.)
layer and nonacosanol tubules
crusts
film and b-diketone
tubules
film
film
film and nonacosanol
tubules
S-layer formation
C-layer formation
C-layer formation
tubules
C-layer formation
C-layer formation
S-layer formation and
tubules
accession
no. BG Bonn
21865
14251
annual plants
9450
19621
19598
14075
annual plants
8047
6521
8077
16296
17084
annual plants
8051
1458
2961
7626
not in culture
2700
Therefore, one image was inverted (negative), adjusted to a matching position
with the second (positive) image in a transparent view, and added using
standard image processing software (Photoline 32, Computerinsel).
(d ) Specimen preparation
The repair of induced voids in epicuticular wax layers was studied on adaxial
(upper) leaf surfaces after removing the original wax layer in small areas (figure 1).
A drop of two-component glue (UHU-plus-Schnellfest 2-Komponentenkleber,
Henkel, Düsseldorf, Germany) was applied onto the leaf surfaces and was removed
after hardening. The areas on which the glue was applied were approximately
3–5 mm in diameter. To obtain a completely clean surface, the wax removal
procedure was repeated twice.
Phil. Trans. R. Soc. A (2009)
Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
1678
K. Koch et al.
cantilever
specimen
microcellulose
hydrophobic coating
specimen holder
piezo
Figure 3. Preparation of fresh, water-containing pieces of plant leaves and stems. Sample holders
were hydrophobized with paraffin to avoid escape of water into the AFM body. Specimens were
fixed on the sample holder with a drop of two-component glue and placed with their cut faces in
wet cellulose fibres to avoid desiccation during investigation.
The Nanoscope IIIa has a quite small specimen chamber, which is designed to
carry samples of approximately 15 mm in diameter. Therefore, two experimental
set-ups of plant preparation were used. The first one is for small leaves that fit
completely or with their apical leaf tips into the specimen chamber. In this
preparation method, the leaf is still connected to the entire plant, which was
placed close to the AFM, as shown in figure 2. The apical parts of the leaf were
mounted on the sample holder plate with the same glue as used for the wax
removal, placing the dewaxed area in the centre. After the second wax removal
procedure, the AFM measurements started immediately, and first images could
be obtained after a few minutes.
AFM investigations of species with larger leaves or stems of succulent plants
required a different preparation technique. The specimen preparation is shown in
figure 3. Pieces of plant leaves or stems were placed with their cut faces in wet
cellulose fibres to avoid desiccation during long-term investigations for several
hours. The cellulose fibres were produced by mechanical crushing of filter paper
(grade 594; Schleicher and Schuell, Dassel, Germany). During the AFM
measurements, a few droplets of water had to be added occasionally to moisten
the cellulose fibres. Specimens were fixed at two edges with a drop of fastdrying glue (UHU-plus-Schnellfest 2-Komponenten Epoxidharz-Kleber, Henkel,
Düsseldorf, Germany) on the sample holder. To avoid escape of water into the
AFM body, the standard metal sample holders were hydrophobized with
paraffin. To avoid drift of the specimen, a reliable mounting with the glue is
important, but nevertheless some drift may occur. If the same position should be
observed for several hours, the sample position needs readjustment.
The investigations have been performed twice with different leaves or shoots.
3. Results and discussion
The new preparation technique provides a water reservoir to keep pieces of
leaves or shoots hydrated. This prevents desiccation and allows observation of
the plant material for 3–5 hours without shrinkage. Therefore, it was possible to
observe the early stages of wax regeneration on the specimen surfaces.
Phil. Trans. R. Soc. A (2009)
Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
Self-healing of voids on plant surfaces
1679
Wax regeneration has been investigated on pieces of the leaves of Citrus limon,
Coffea arabica, Iris germanica and Thalictrum flavum glaucum. Three succulent
plants, whose intact leaves (Kalanchoe daigremontianum, Lithops turbiniformis)
or shoots (Aporocactus flagelliformis) were too thick for fixation in the AFM
specimen chamber, were investigated by using the described new preparation
technique. The observed wax regeneration patterns are listed in table 1. The
modes of wax regeneration are discussed in §3a. In the other 10 species listed in
table 1, wax regeneration was investigated on intact leaves. Large differences
were found in the rate of wax regeneration, and two modes of wax layer
formation were found. These differences are not related to the kind of specimen
preparation. The study here focuses on the early stages of wax film formation on
plant surfaces. However, in some species, the regeneration of three-dimensional
wax crystals starts parallel to wax film formation (table 1).
(a ) Concentric and stripe-shaped formation of wax films
Two different modes of wax layer formation, a concentric (planar) and a
stripe-shaped (linear) formation of wax films, were found and are introduced here
with representative species as examples. A concentric layer formation was
observed for 11 species, indicated in table 1 as C-layer formation. Within these
species, large differences were found in the amount of newly formed wax terraces
(density of wax layer nuclei) and in the growth rate of the wax layer formation.
In figure 4, the formation of a monomolecular layer of wax on a leaf of the
succulent plant L. turbiniformis is shown. Figure 4a–c shows the increase in the
wax layer at 12, 27 and 38 min after wax removal. The appearing wax forms
almost concentric (C-layer) terraces, and the growth of these layers occurs by the
addition of new wax at the outermost edges of the layers, as shown in figure 4d,
which has been produced by overlaying and subtraction of figure 4b,c. Thus,
figure 4d shows the wax added within 11 min.
In one investigation of wax regeneration of Taxus baccata, the removal of the
original wax layer was incomplete, but in this initial situation it could be
observed how regenerating wax films fill up the wax-free voids on the cuticle. The
image series in figure 5 shows some thicker layers, which are the wax residues,
and, in the centre between the residues, a growing new wax layer. Figure 5a
represents the wax regeneration 23 min after partial removal of the original wax
layer, figure 5b has been recorded after 33 min, figure 5c after 70 min and
figure 5d after 138 min. The nearly concentric growth of the new wax layer
changes its growth directions when it comes into contact with non-growing wax
residues (figure 5c), and thereby effectively refills the spaces between the already
existing wax residues. The continuous growth of the wax film occurs by a process
of self-assembly.
In a second mode of layer formation, observed in seven species, the new wax
material grows in a stripe-shaped pattern. Species with this type of layer
formation are indicated in table 1 as S-layer formation. Ipheion uniflorum is the
only species for which both modes of wax film formation have been observed. One
representative species in which S-layer formation is shown is Lathyrus odoratus.
The AFM images in figure 6a–c show that the stripe-shaped layers grew in
different directions. Figure 6a has been recorded 109 min, figure 6b at 125 min
and figure 6c at 149 min after removal of the wax. When growing layers come
Phil. Trans. R. Soc. A (2009)
Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
1680
K. Koch et al.
(a)
1 µm
(b)
1 µm
(c)
1 µm
(d)
1 µm
Figure 4. Concentric wax layer formation on a leaf of L. turbiniformis over 38 min. (a) The wax
regeneration 12 min after removal of the original wax layer, (b) after 27 min and (c) after 38 min.
Growth of the wax film occurs by the addition of new wax at the outermost edges of the concentric
wax circles, demonstrated by (d ), which has been produced by overlaying of (b) and (c) and
subtraction of (c) from (b). (d ) The wax added within 11 min.
into contact with already existing layers, they start to overgrow them. The
lateral sides of the stripes accumulate new wax material, as indicated by the
closure of the hollows formed between the stripes, but, for a rapid re-covering of
the cuticle with a complete wax layer, this kind of wax regeneration seems to be
less efficient. Growth of these S-layers occurs by the addition of new wax
preferentially at the edges of the terminal ends of the stripes, demonstrated
by figure 6d, which shows the amount of new wax accumulated within 16 min.
Figure 6d has been produced by overlaying of figure 6a,b and subtraction of
figure 6a from b.
(b ) Formation of multilayered wax films
Multilayer wax formation has been described for Galanthus nivalis (Koch et al.
2004) and Euphorbia lathyris (Dommisse 2007). In these studies, it has been
shown that the wax films on plant surfaces are built by several molecular layers.
Phil. Trans. R. Soc. A (2009)
Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
1681
Self-healing of voids on plant surfaces
(a)
(b)
1 µm
(c)
1 µm
(d)
1 µm
1 µm
Figure 5. AFM image series of the wax regeneration on a leaf of T. baccata. (a) The wax
regeneration 23 min after partial removal of the original wax layer, (b) after 33 min, (c) after 70 min
and (d ) after 138 min. (c) The nearly concentric growth of the new wax layer located in the centres
of the figures changes its growth directions when it comes into contact with non-growing
wax residues.
AFM height measurements at the edges of the growing layers represent the
heights of vertically orientated long-chain hydrocarbons, with a chain length
between C20 and C40. In this study, the average thickness of the wax layers of
L. odoratus (figure 6) is 8 nm and represents a bilayer formation of aliphatic
hydrocarbon molecules of approximately 30–32 carbon atoms. In T. baccata
(figure 5d ), the average heights of the steps in the wax layers are 6.5, 9.0 and
12.1 nm, indicating that the wax film is composed of two, three and four layers of
molecules with chain lengths of approximately 3 nm (C24–C26 molecules). In
L. turbiniformis (figure 4), the average heights of the wax layers are 4.2 and
6 nm, representing the chain length of vertically orientated long-chain
hydrocarbons, with a chain length of approximately 32 and 46 carbon atoms.
In I. uniflorum (figure 6), an independent development of two different layers was
observed. While a ‘normal’ stripe-shaped layer grew slowly, a layer with variable
thickness (from 3 to 12 nm) spread over the observed area in less than 1 hour.
Phil. Trans. R. Soc. A (2009)
Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
1682
K. Koch et al.
(a)
(b)
1 µm
(c)
1 µm
(d)
1 µm
1 µm
Figure 6. The AFM images show the stripe-like formation of wax layers (S-layer formation) on
L. odoratus: (a) 109 min, (b) 125 min and (c) 149 min after removal of the wax. Growth of these
layers occurs by the addition of new wax material mainly at the edges of the terminal ends of the
stripes. This is demonstrated in (d ), which shows the part of the new wax accumulated within
16 min. It has been produced by overlaying of (a) and (b) and subtraction of (b) from (a).
Probably one of the layers grows under an already existing wax layer, the other
one on top of it. During the formation of multilayered wax films and for the
formation of three-dimensional wax crystals on the films, diffusion of new wax
material through already existing wax layers is assumed (Koch et al. 2004).
However, in I. uniflorum, already existing wax structures were pushed up by a
new layer of regenerated wax below. This process of wax formation is shown in
figure 7. Here, a formation of S-layers, denoted in figure 7a by the upper arrow,
and a fast growing layer below, denoted by the lower arrow, is shown. Height
measurements made at the edge of this layer show a step height between 3 and
12 nm, which indicates that this layer is composed of more than one molecular
layer. Figure 7b, recorded only 4 min later, shows that the wax layer has grown
underneath the S-layer structures. In figure 7c, 44 min after the original wax has
been removed, the wax layer has covered nearly 80 per cent of the scanned area,
whereas the S-layers grew very slowly. After a total time of 53 min (figure 7d ),
the scanned surface area is completely covered with the new wax layer. Even the
higher structure in the cuticle, visible at the right side of the figures, was
overgrown by the new layer.
Phil. Trans. R. Soc. A (2009)
Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
1683
Self-healing of voids on plant surfaces
(a)
(b)
1 µm
(c)
1 µm
(d)
1 µm
1 µm
Figure 7. AFM images of a multilayer wax film formation on I. uniflorum. In (a), recorded 32 min
after removal of the original waxes, the formation of S-layer film is denoted by the upper arrow.
The edge of a second, rapidly growing layer is denoted by the lower arrow. In (b), only 4 min
later, the wax layer has grown underneath the S-layer structures. In (c), recorded another 4 min
later, the wax layer has covered nearly 80% of the scanned area, whereas the S-layers grew very
slowly. (d ) After a total time of 53 min, the scanned surface area is completely covered with the
new wax layer.
The data presented here show that all 17 species started to regenerate a wax film
immediately after removal of the wax, but differences in the intensity of wax
regeneration were found, and in some investigations no wax regeneration could be
observed (data not shown). The fact that some species showed no wax regeneration
is a strong indication that wax regeneration is not based on pure diffusion of
intracuticular wax to the surface. If this were the case, on each specimen at least a
few layers of wax would have been found. The ability of self-healing seems to
correlate with the new production of wax. Wax synthesis is generally high during
the development of the leaves (Baker 1982; Post-Beitenmiller 1996). Thus it can be
assumed that the lower intensity of wax regeneration found in some older leaves is
mainly based on differences of wax synthesis. Differences in wax regeneration in
leaves of different ages have been found by SEM studies (Neinhuis et al. 2001).
Phil. Trans. R. Soc. A (2009)
Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
1684
K. Koch et al.
In this study, mature leaves from 6 out of 16 species showed no wax regeneration,
whereas 13 of 16 species showed a high intensity of wax regeneration when leaves
were in development. However, external factors such as changes in temperature and
air humidity can also influence the dynamics of wax transport through the plant
cuticle (Schreiber & Schönherr 1993; Schreiber 2001) and the epicuticular
movement of the wax molecules.
4. Mechanisms of wax formation
(a ) C- and S-layer formations of wax films
Two modes of wax film formation have been observed: concentric (C) layer
formation, in which the new wax spreads out in a concentric or planar way; and
stripe-shaped (S) layer formation, in which the wax film grows in the form of
stripes or lines. Both growth modes are a process of molecular self-assembly,
in which new wax molecules are preferentially added to the edges of existing
layers, resulting in a layer structure. The stripe-like wax layer results from a
faster accumulation of new material at the terminal ends of the stripes, whereas
in the concentric growing wax layers the new wax is added approximately
equally onto all sides of the layer. When the layer growth continues, a complete
monolayer, as schematically shown in figure 8a, is formed, and, later, a multilayered wax film is formed.
Two factors can induce differences in C- and S-layer formation. One is
differences in the chemical composition of the wax and the other is the unknown
molecular structure of the underlying cuticle. In most species, the wax film
occurs together with three-dimensional wax crystals, but until now it has not
been possible to separate both structures for separate chemical analysis. The
AFM images show that the regenerated wax layer thicknesses vary around 4 nm
(the average molecule length of typical aliphatic wax compounds with a chain
length of 30 carbon atoms). The mass of a single molecular wax layer, as
observed with the AFM, is not sufficient for a chemical analysis by gas
chromatography and/or mass spectroscopy. Thus the general lack of knowledge
about the chemistry of the thin wax film makes an interpretation difficult. By
comparison, a 1 mm thick wax layer on a leaf consists of approximately 250
molecular layers of wax. Current knowledge about the chemistry of the
underlying wax films is based on conclusions made from recrystallization
experiments with waxes and single wax compounds. For wax tubules and some
platelets, it has been shown that only one or two compounds of the wax mixtures
are responsible for the formation of the three-dimensional structures (Jeffree
2006; Koch & Ensikat 2008). As a consequence, it is assumed that the majority of
wax compounds are located in the wax film.
For some species where only wax films and no three-dimensional waxes exist,
chemical data are known. A well-investigated species is Prunus laurocerasus,
which showed a C-layer formation. Jetter & Schäffer (2001) showed that these
films are composed of approximately 39 different aliphatic wax compounds, but
they also gave evidence that the chemical composition varies during the
ontogeny of the leaves. Thus, the next step for understanding the differences in
wax film formation could be in vitro recrystallization of the single wax
components and their mixtures.
Phil. Trans. R. Soc. A (2009)
Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
Self-healing of voids on plant surfaces
1685
(a)
(b)
(c)
Figure 8. Layer formation and formation of multilayered wax films on plant surfaces. Newly added
wax molecules are drawn in dark. In (a), the formation of a monolayer of wax molecules is based on
the addition of new molecules at the edges of the existing wax. The formation of multilayers is
schematically shown in (b) and (c). In (b), new wax molecules diffuse through the already existing
layers, whereas, in (c), the existing wax layers are lifted up by the new wax layer below.
(b ) Formation of multilayered wax films
The mechanisms of formation and growth of plant waxes have been discussed
for many years. Since the first AFM analysis of wax regeneration (Koch et al.
2004), further in vitro studies of wax crystal formation have been carried out
Phil. Trans. R. Soc. A (2009)
Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
1686
K. Koch et al.
(Koch et al. 2006a,b). Based on these and the data presented here, it can be
concluded that two mechanisms of layer formation lead to multilayered wax
films, as schematically shown in figure 8b,c.
In the first mode (figure 8b), new wax material moves through the existing,
complete wax layers. With only two exceptions (discussed below), this mode was
found in all species after the first layer of waxes was completely regenerated.
However, this mode predicts a certain degree of permeability of the already existing
wax layers to provide the wax molecules necessary for the growth of threedimensional waxes on the wax films. Schreiber & Schönherr (1993) performed
transport studies with reconstituted wax layers and showed that aliphatic
molecules are mobile within the wax mixture. Reynhardt & Riederer (1994)
emphasized the importance of crystalline and amorphous zones in plant waxes for
the diffusion of molecules through the cuticle. Reynhard (1997) demonstrated that a
mixture of short and long chains form amorphous zones of higher fluidity. These
amorphous zones could be the pathways for the new wax molecules on their way
through a wax film. However, the molecule mobility, and therefore the intensity of
wax regeneration, can be reduced when the wax film is in a stable crystallized form.
In the second case, schematically shown in figure 8c, existing waxes are lifted up
by the growing new wax layer underneath. This kind of multilayer formation has been
observed only during wax regeneration in two plants, I. uniflorum and T. baccata.
The I. uniflorum AFM micrographs demonstrate that existing striped wax layers
were not influenced by the much faster growing second wax layer. Compared with
other species, these species showed a very fast regeneration of the wax layer, and it
can only be speculated that the amount of new wax that appeared on the cuticle
surface was too great to diffuse quickly through the already existing wax layers.
5. Conclusions
The preparation technique for AFM for small pieces of living plant material,
described in this paper, provides the opportunity to study plant surface structures,
or wax regeneration, over several hours with minimized material shrinkage by
water loss. The comparative study of wax film formation in 17 different species
showed two different kinds of wax film formation, described as concentric layer
formation and striped layer formation. The AFM examinations presented here
showed that regeneration of epicuticular wax films on living plant surfaces is a
highly dynamic and comparatively fast process, reflecting the importance of
a continuous wax coverage of leaves. This study showed that most plants are able
to refill voids within the epicuticular wax layer. The process of wax film formation
occurs by self-assembly of the wax molecules into layered structures, but it seems to
be limited in some plants by non-existing or low wax synthesis inside the cells. The
self-healing process found in plants might be an interesting system for the
development of coatings of materials that are permeable for small molecules.
In future, AFM investigations of recrystallization of isolated film-forming waxes
might give a deeper understanding of the correlation between the chemistry and the
observed different modes of layer formation.
The authors thank the German Science Foundation (Deutsche Forschungsgemeinschaft), Deutsche
Bundesstiftung Umwelt (DBU) and the Bundesministerium für Bildung und Forschung (BMBF)
for the financial support of their research. Special thanks go to Dr H. Bargel for his comments and
discussions during the preparation of the manuscript.
Phil. Trans. R. Soc. A (2009)
Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
Self-healing of voids on plant surfaces
1687
References
Anton, L. H., Ewers, F. W., Hammerschmidt, R. & Klomparens, K. L. 1994 Mechanisms of
deposition of epicuticular wax in leaves of broccoli, Brassica oleracea L. var. capitata L. New
Phytol. 26, 505–510. (doi:10.1111/j.1469-8137.1994.tb04248.x)
Baker, E. A. 1982 Chemistry and morphology of plant epicuticular waxes. In The plant cuticle (eds
D. F. Cutler, K. L. Alvin & C. E. Price), pp. 139–165. London, UK: Academic Press.
Barnes, J. D. & Cardoso-Vilhena, J. 1996 Interactions between electromagnetic radiation and the
plant cuticle. In Plant cuticles, an integrated approach (ed. G. Kerstiens), pp. 157–170. Oxford,
UK: BIOS Scientific Publishers.
Barthlott, W. & Neinhuis, C. 1997 The purity of sacred lotus or escape from contamination in
biological surfaces. Planta 202, 1–8. (doi:10.1007/s004250050096)
Barthlott, W., Neinhuis, C., Cutler, D., Ditsch, F., Meusel, I., Theisen, I. & Wilhelmi, H. 1998
Classification and terminology of plant epicuticular waxes. Bot. J. Linn. Soc. 126, 237–260.
(doi:10.1006/bojl.1997.0137)
Buchholz, A. & Schönherr, J. 2000 Thermodynamic analysis of diffusion of non-electrolytes across
plant cuticles in the presence and absence of the plasticiser tributyl phosphate. Planta 212,
103–111. (doi:10.1007/s004250000372)
Dommisse, A. 2007 Self-assembly and pattern formation of epicuticular waxes on plant surfaces.
Dissertation, Rheinische Friedrich-Wilhelms Universität Bonn.
Ensikat, H. J., Neinhuis, C. & Barthlott, W. 2000 Direct access to plant epicuticular wax crystals
by a new mechanical isolation method. Int. J. Plant Sci. 161, 143–148. (doi:10.1086/314234)
Ensikat, H. J., Boese, M., Mader, W., Barthlott, W. & Koch, K. 2006 Crystallinity of plant
epicuticular waxes: electron and X-ray diffraction studies. Chem. Phys. Lipids 144, 45–59.
(doi:10.1016/j.chemphyslip.2006.06.016)
Gorb, E., Haas, K., Henrich, A., Enders, S., Barbakadze, N. & Gorb, S. 2005 Composite structure
of the crystalline epicuticular wax layer of the slippery zone in the pitchers of the carnivorous
plant Nepenthes alata and its effect on the insect attachment. J. Exp. Biol. 208, 4651–4662.
(doi:10.1242/jeb.01939)
Jeffree, C. E. 2006 The fine structure of the plant cuticle. In Biology of the plant cuticle (eds
M. Riederer & C. Müller), pp. 11–125. Oxford, UK: Blackwell.
Jeffree, C. E., Baker, E. A. & Holloway, P. J. 1975 Ultrastructure and recrystallization of plant
epicuticular waxes. New Phytol. 75, 539–549. (doi:10.1111/j.1469-8137.1975.tb01417.x)
Jetter, R. & Schäffer, S. 2001 Chemical composition of Prunus laurocerasus leaf surface. Dynamic
changes of the epicuticular wax film during leaf development. Plant Physiol. 126, 1725–1737.
(doi:10.1104/pp.126.4.1725)
Koch, K. & Ensikat, H. J. 2008 The hydrophobic coatings of plant surfaces: epicuticular wax
crystals and their morphologies, crystallinity and molecular self-assembly. Micron 39, 759–772.
(doi:10.1016/j.micron.2007.11.010)
Koch, K., Neinhuis, C., Ensikat, H. J. & Barthlott, W. 2004 Self assembly of epicuticular waxes on
plant surfaces investigated by atomic force microscopy (AFM). J. Exp. Bot. 55, 711–718.
(doi:10.1093/jxb/erh077)
Koch, K., Barthlott, W., Koch, S., Hommes, A., Wandelt, K., Mamdouh, W., De-Feyter, S. &
Broekmann, P. 2006a Structural analysis of wheat wax (Triticum aestivum): from the molecular
level to three dimensional crystals. Planta 223, 258–270. (doi:10.1007/s00425-005-0081-3)
Koch, K., Dommisse, A. & Barthlott, W. 2006b Chemistry and crystal growth of plant wax tubules
of lotus (Nelumbo nucifera) and nasturtium (Tropaeolum majus) leaves on technical substrates.
Cryst. Growth Des. 6, 2571–2578. (doi:10.1021/cg060035w)
Koch, K., Bhushan, B. & Barthlott, W. 2008 Diversity of structure, morphology and wetting of
plant surfaces. Invited review. Soft Matter 4, 1943–1963. (doi:10.1039/b804854a)
Koch, K., Bhushan, B. & Barthlott, W. 2009 Multifunctional surface structures of
plants: an inspiration for biomimetics. Prog. Mater. Sci. 54, 137–178. (doi:10.1016/j.pmatsci.
2008.07.003)
Phil. Trans. R. Soc. A (2009)
Downloaded from http://rsta.royalsocietypublishing.org/ on July 31, 2017
1688
K. Koch et al.
Neinhuis, C., Koch, K. & Barthlott, W. 2001 Movement and regeneration of epicuticular waxes
through plant cuticles. Planta 213, 427–434. (doi:10.1007/s004250100530)
Popp, C., Burghardt, M., Friedmann, A. & Riederer, M. 2005 Characterization of hydrophilic and
lipophilic pathways of Hedera helix L. cuticular membranes: permeation of water and uncharged
organic compounds. J. Exp. Bot. 56, 2797–2806. (doi:10.1093/jxb/eri272)
Post-Beitenmiller, D. 1996 Biochemistry and molecular biology of wax production in plants. Annu.
Rev. Plant Physiol. Plant Mol. Biol. 47, 405–430. (doi:10.1146/annurev.arplant.47.1.405)
Reynhard, E. C. 1997 The role of hydrogen bonding in the cuticular wax of Hordeum vulgare L.
Eur. Biophys. J. 26, 195–201. (doi:10.1007/s002490050071)
Reynhardt, E. C. & Riederer, M. 1994 Structures and molecular dynamics of plant waxes. II.
Cuticular waxes from leaves of Fagus sylvatica L. and Hordeum vulgare L. Eur. Biophys. J. 23,
59–70. (doi:10.1007/BF00192206)
Riederer, M. & Schreiber, L. 1995 Waxes—the transport barriers of plant cuticles. In Waxes:
chemistry, molecular biology and functions (ed. R. J. Hamilton), pp. 131–156. Dundee, UK: The
Oily Press.
Riederer, M. & Schreiber, L. 2001 Protecting against water loss: analysis of the barrier properties
of plant cuticles. J. Exp. Bot. 52, 2023–2032. (doi:10.1093/jexbot/52.363.2023)
Schönherr, J. 2006 Characterization of aqueous pores in plant cuticles and permeation of ionic
solutes. J. Exp. Bot. 57, 2471–2491. (doi:10.1093/jxb/erj217)
Schreiber, L. 2001 Effect of temperature on cuticular transpiration of isolated cuticular membranes
and leaf discs. J. Exp. Bot. 52, 1893–1900. (doi:10.1093/jexbot/52.362.1893)
Schreiber, L. & Schönherr, J. 1993 Mobilities of organic compounds in reconstituted cuticular wax
of barley leaves: determination of diffusion coefficients. Pestic. Sci. 38, 353–361. (doi:10.1002/
ps.2780380413)
Phil. Trans. R. Soc. A (2009)