Chemical Composition of the Prunus laurocerasus Leaf
Surface. Dynamic Changes of the Epicuticular Wax
Film during Leaf Development1
Reinhard Jetter* and Stefanie Schäffer
Julius-von-Sachs-Institut für Biowissenschaften, Lehrstuhl für Botanik II, Universität Würzburg, Julius-vonSachs-Platz 3, D–97082 Würzburg, Germany
The seasonal development of adaxial Prunus laurocerasus leaf surfaces was studied using newly developed methods for the
mechanical removal of epicuticular waxes. During epidermal cell expansion, more than 50 ␮g leaf⫺1 of alkyl acetates
accumulated within 10 d, forming an epicuticular wax film approximately 30 nm thick. Then, alcohols dominated for 18 d
of leaf development, before alkanes accumulated in an epicuticular wax film with steadily increasing thickness (approximately 60 nm after 60 d), accompanied by small amounts of fatty acids, aldehydes, and alkyl esters. In contrast, the
intracuticular waxes stayed fairly constant during development, being dominated by triterpenoids that could not be detected
in the epicuticular waxes. The accumulation rates of all cuticular components are indicative for spontaneous segregation of
intra- and epicuticular fractions during diffusional transport within the cuticle. This is the first report quantifying the loss
of individual compound classes (acetates and alcohols) from the epicuticular wax mixture. Experiments with isolated
epicuticular films showed that neither chemical conversion within the epicuticular film nor erosion/evaporation of wax
constituents could account for this effect. Instead, transport of epicuticular compounds back into the tissue seems likely.
Possible ecological and physiological functions of the coordinate changes in the composition of the plant surface layers are
discussed.
Leaves of higher plants are covered by a cuticle, i.e.
an extracellular membrane consisting of a polymeric
cutin matrix and soluble cuticular waxes. A specific
portion of the waxes is embedded within the polymer
framework (Holloway, 1982) and should be designated as “intracuticular waxes” (Walton, 1990). It is
widely accepted that all plant cuticles also carry a
thin film of “epicuticular waxes” on the surface of
their cutin matrix (Baker, 1982). In addition, on particular plant species and organs the surface waxes
can form microscopic aggregates, “epicuticular wax
crystals,” protruding from the wax film (Barthlott et
al., 1998).
Due to its position at the interface between the
plant and its atmospheric environment, the cuticle
performs multiple physiological and ecological roles.
To be more specific, some of the most important
properties of the cuticle must be determined by epicuticular wax films because they are located at its
very surface. On one hand, there is circumstantial
evidence that the most effective part of the cuticular
transpiration barrier resides in waxes at or near the
cuticle surface (Schönherr, 1976; Schönherr and Riederer, 1988). On the other hand, this outermost cuticular layer must necessarily be relevant for host
1
This work was supported by the Fonds der Chemischen Industrie (grant) and by the Deutsche Forschungsgemeinschaft
(grant no. Sonderforschungsbereich 567 “Mechanisms of Interspecific Interactions of Organisms”).
* Corresponding author; e-mail [email protected];
fax 49 –931– 888 – 6235.
recognition by fungi as well as insects. It has been
shown that many pathogens and herbivores probe
the chemical nature of their substrate (Schoonhoven
et al., 1998). Wax constituents act as allelochemicals
by influencing fungal development (Carver et al.,
1990; Podila et al., 1993; Flaishman et al., 1995)
and/or insect behavior (Städler, 1986; van Loon et al.,
1992). To further understand the molecular mechanisms determining these essential functions of plant
surfaces, epicuticular wax compositions must be documented with adequate spatial and temporal
resolution.
In the past, surface extraction with organic solvents
was routinely employed to probe cuticular waxes for
chemical analysis (Holloway, 1984). Because solvent
molecules release both intra- and epicuticular waxes
together, only the overall composition of the complete wax mixture could be assessed (Walton, 1990).
This bulk wax composition was usually assumed to
reflect the surface composition, ignoring possible differences between intra- and epicuticular waxes.
Meanwhile, some evidence for chemical differences
between intra- and epicuticular waxes has emerged.
First, comparative chemical and morphological investigations showed that special wax constituents
form the epicuticular crystals on diverse plant surfaces (Jeffree et al., 1975; Gülz et al., 1992; Jetter and
Riederer, 1994; Jetter and Riederer, 1999; Meusel et
al., 1999; Markstädter et al., 2000). In these cases
individual constituents are accumulated at the very
plant surface (Jeffree et al., 1975; Baker, 1982), prob-
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Jetter and Schäffer
ably by diffusion and spontaneous phase separation
of compounds. Second, for those plant surfaces covered by surface wax films without epicuticular crystals, a distinction between intra- and epicuticular
constituents was attempted by either mechanically
stripping (Haas and Rentschler, 1984) or chemically
washing (Silva Fernandes et al., 1964; Holloway,
1974; Baker and Procopiou, 1975; Baker et al., 1975;
Svenningsson, 1988; Garrec et al., 1995) the film off
the plant surface. None of these methods had sufficient selectivity to unambigously localize individual
compounds in either cuticular sublayer. However, in
all cases transverse gradients in the percentages of
individual compounds suggested chemical differences between intracuticular waxes and the surface
film. Taken together, all these studies clearly indicated that surface composition cannot be extrapolated from extractive bulk wax mixtures.
First methods were devised only recently allowing
the selective preparation of epicuticular wax films
and hence the direct analysis of surface compositions
(Ensikat et al., 2000; Jetter et al., 2000). For Prunus
laurocerasus leaves, a layered arrangement of wax
fractions was revealed, with aliphatic constituents
concentrated in the epicuticular film and pentacyclic
triterpenoids located exclusively in the intracuticular
compartment. In these experiments, transverse gradients within the cuticular components were quantified for the first time. However, because wax preparation included freeze-thaw cycles of the leaf tissue
the methods could not be applied in vivo to study the
dynamics of cuticular wax layers. Crucial aspects of
cuticle formation, especially the mechanisms and
regulation of surface wax arrangement, hence remained unknown. As a consequence, we now initiated in vivo investigations into the seasonal development of epicuticular waxes of P. laurocerasus. The
main objective of the present work was to assess: (a)
the rates for the accumulation of individual compound classes at the leaf surface, (b) the processes
leading to segregation of intra- and epicuticular wax
fractions, and (c) the mechanisms explaining the net
loss of epicuticular wax constituents. To investigate
all these aspects of cuticle dynamics, new procedures
had to be developed employing adhesives to selectively probe epicuticular waxes in situ.
through C36 alcohols, C23 through C36 aldehydes, C20
through C34 fatty acids, and C22 through C36 acetates
(Fig. 1A). Diverse other adhesive materials were now
tested as alternatives to remove surface compounds
from the leaves.
Various glues yielded wax mixtures similar to
those of cryo-adhesive treatments, but the overall
amount, pattern of individual compound classes, and
chain length distributions were best reproduced with
gum arabic. Repeated treatments of a given surface
with this polymer released mixtures of similar composition (Fig. 1A) but with drastically decreasing
yields. In none of the mechanically prepared wax
samples triterpenoids could be detected. Only when
the gum arabic-treated leaf discs subsequently were
extracted with chloroform were two triterpenoids,
ursolic and oleanolic acid, released (Fig. 1B). Besides,
small amounts of the aliphatic compound classes
previously identified in gum arabic preparations
could be detected in the solvent extracts. The total
amounts both of individual constituents and of compound classes removed by three successive gum arabic treatments plus subsequent chloroform extraction were equal to their yields in a single extraction of
authentic surfaces (Fig. 1B).
RESULTS
The first objective of the present investigation was
to develop methods for selectively probing epicuticular wax films in situ. It had previously been shown
that adhesives can be employed to mechanically remove surface waxes for chemical analysis (Jetter et
al., 2000). The most selective method employing frozen glycerol as cryo-adhesive was used to generate
reference data on the epicuticular wax composition
of adult P. laurocerasus leaves. This yielded a characteristic pattern of C25 through C35 alkanes, C21
1726
Figure 1. Composition of wax mixtures removed from adaxial surfaces of mature P. laurocerasus leaves by adhesive treatment or
solvent extraction. Yields of individual compound classes (␮g cm⫺2)
are given as mean values (n ⫽ 6) with SD. A, Surface waxes were
sampled in a single treatment employing frozen glycerol as a cryoadhesive or in three consecutive treatments with an adhesive polymer film formed by an aqueous solution of gum arabic. B, Comparison between wax mixtures removed by repeated gum arabic
treatments, released by successive chloroform extraction of the same
surface, or by extraction of the native surface (without prior mechanical treatment).
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Plant Physiol. Vol. 126, 2001
Leaf Epicuticular Wax Composition of Prunus laurocerasus
Further experiments showed that gum arabic could
be left for up to 10 d on P. laurocerasus leaves without
causing visible damage. The polymer could also be
applied to and removed from immature leaves without disturbing their development. Hence, a detailed
investigation using gum arabic to study the seasonal
development of adaxial leaf surfaces was initiated.
The polymer was employed in situ to remove the
epicuticular wax film from the entire surface of
leaves in the earliest accessible stage of development.
This was first possible 3 d after bud break and thus
would define the starting point in the time course.
The initially removed epicuticular waxes contained
high percentages of acetates with small admixtures of
alkanes, alcohols, and fatty acids (Table I). Homolog
distributions ranged from C20 to C36 with a predominance of chain lengths C24 and C26. The total yield of
removed epicuticular compounds corresponded to
an original coverage of 4.0 ⫾ 0.94 ␮g cm⫺2.
Leaf development was monitored for 57 d after the
first treatment with gum arabic. During the first 10 d
leaf surface areas increased linearly with continuously growing variability (Fig. 2A, dotted line). In the
same time interval, a steady decrease in the density
of epidermal cells per unit area was observed (Fig.
2B) and the total number of cells per leaf (calculated
from data in Fig. 2, A and B) remained constant at
approximately 3.6 106 (Fig. 2C). This first phase of the
time course thus was characterized by expansive
growth of epidermal cells. During the ensuing time
interval (d 13–57) the leaf surface area, epidermal cell
density and total cell numbers remained approximately unchanged (Fig. 2). In the developmental investigation of surface waxes, the chemical data from
all time points had to be compared and therefore
were normalized using standard leaf areas. These
were calculated by performing a linear regression for
the phase of cell expansion (Ad ⫽ 0 ⫽ 5.8 cm2; ⌬A ⫽
2.2 cm2 d⫺1) and by averaging the final area of all
individual leaves (A ⫽ 29.1 cm2; Fig. 2A, bold lines).
This procedure helped to eliminate small fluctuations
in some of the values, especially at d 30 and 43, that
were caused by overrepresentation of certain leaf
batches in respective samples. Because the six individual leaves sampled and analyzed at d 30 and 43
mostly belonged to batch E (latest bud break), they
had relatively small final areas (Fig. 2A) and epidermal cell numbers (Fig. 2C). For all individual leaves
in the present investigation, a strict correlation was
found between the date of bud break, the time before
they reached full size, and their final surface area.
Development of epicuticular wax coverage and
composition during a period of 57 d after the first
wax removal was studied. For this purpose, at each
sampling point six leaves were selected and their
surface waxes were probed with a second gum arabic
treatment and analyzed. Total yields of epicuticular
waxes per unit area rose steeply from 0 to approximatley 3 ␮g cm⫺2 in the 5 d after the first removal. In
the ensuing time interval to d 57, a smooth overall
increase to 6 ␮g cm⫺2 could be seen. A drastic rise
and loss of coverages around d 23 transiently was
observed before moderate increase was resumed. Because the leaf areas remained constant over most of
the investigated period, the epicuticular wax
amounts per leaf were very similiar to the development of coverages per unit area. Hence, changes in
wax composition can be bilanced on a whole-leaf
basis without loss of information.
In the development of adaxial surface waxes of P.
laurocerasus leaves, different phases could be distinguished according to the amounts of individual com-
Table I. Composition of epicuticular waxes on adaxial surfaces of P. laurocerasus leaves 3 d after bud break
Mean values (n ⫽ 20) and SD are given for the homologous composition (% of the fraction) and the coverage (␮g/cm⫺2) of compound classes.
a
Chain Length
Alkanes
Alcohols
Fatty Acids
Alkyl Acetates
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Coverage
–
–
–
–
–
–
–
100.0 ⫾ 0
–
tra
–
tr
–
–
–
–
–
0.03 ⫾ 0.01
–
–
3.4 ⫾ 1.5
1.0 ⫾ 0.7
40.0 ⫾ 4.3
3.3 ⫾ 1.4
22.5 ⫾ 2.1
0.5 ⫾ 0.5
9.8 ⫾ 1.2
–
13.1 ⫾ 2.0
–
tr
–
2.7 ⫾ 2.5
–
3.8 ⫾ 2.3
0.19 ⫾ 0.08
1.4 ⫾ 0.9
–
4.3 ⫾ 1.5
0.7 ⫾ 0.4
53.9 ⫾ 6.1
3.6 ⫾ 1.8
31.5 ⫾ 5.3
tr
1.6 ⫾ 0.9
0.6 ⫾ 0.9
1.3 ⫾ 0.7
–
1.3 ⫾ 0.7
–
–
–
–
0.16 ⫾ 0.11
–
–
1.2 ⫾ 0.4
0.2 ⫾ 0.1
31.8 ⫾ 3.4
4.4 ⫾ 0.7
36.1 ⫾ 1.7
1.5 ⫾ 0.4
11.4 ⫾ 0.7
0.6 ⫾ 0.1
6.7 ⫾ 0.5
0.3 ⫾ 0.1
3.5 ⫾ 1.0
0.1 ⫾ 0.1
1.6 ⫾ 0.5
–
0.7 ⫾ 0.2
3.62 ⫾ 0.74
tr, Traces; i.e. less than 0.5% in the fraction detectable.
Plant Physiol. Vol. 126, 2001
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Jetter and Schäffer
Figure 2. Size development of P. laurocerasus leaves. Averages and
SD (n ⫽ 6) for the surface areas (A), the epidermal cell densities (B),
and the calculated (approximate) epidermal cell numbers (C) are
given for all the leaves sampled in the present investigation. Straight
lines in A show the average area values used for normalizing wax
coverage data.
pound classes present (Fig. 3). First, a steady increase
in the absolute quantities of acetates during 8 d after
the initial wax removal resulted in a strong predominance of this compound class. In the course of the
ensuing development, the total amounts of acetates
on the adaxial surface steadily declined. In a converse manner, the quantities of alcohols steadily increased between d 8 and 23, at first compensating
and then exceeding the loss of acetates. Total leaf
coverages consequently leveled off beween d 8 and
13 before they reached a maximum at d 23. Due to
losses of the predominating alcohols, the total wax
amounts were again diminished in the following
phase. Only after d 30 an increase in the absolute
quantities of alkanes could compensate for further
deprivation of both alcohols and acetates, and the
epicuticular wax mixture was then dominated by
alkanes. The aldehydes and alkyl esters, although at
lower amounts, showed developments similar to the
alkanes, whereas the time course of fatty acid quantities paralleled that of the alcohols.
It should be noted that an epicuticular wax layer
that was chemically indistinguishable from the original film (Table I) accumulated within 2 d after the
1728
initial wax removal (Fig. 3). This early increase of
waxes could either reflect the normal development or
a restoration of the disturbed surface. To distinguish
between these possibilities, the epicuticular film was
investigated on control leaves without initial wax
removal. Gum arabic treatment at d 5 of the present
time frame yielded 54 ␮g cm⫺2 of wax containing
91% (w/w) alcohol acetates. In a similar manner, wax
amounts and composition at d 8, 10, and 57 were
identical to those on leaves from which waxes had
originally been removed (data not shown). This
shows that the normal ontogenetic development of
epicuticular waxes had been resumed within 5 d after
the initial wax removal. Hence, in Figure 3 chemical
data for d 1 and 3 reflect epicuticular wax regeneration, whereas those for d ⱖ 5 characterize the normal
surface ontogeny.
The chain length distribution of compounds within
the major fractions of P. laurocerasus waxes changed
drastically during development (Fig. 4). Acetates, alcohols, and fatty acids showed a clear predominance
of the C24 homologs with admixtures of C26 compounds at d 8. In contrast, after d 23 the acetate
fraction had a homolog pattern centered around C32.
The chain length distribution of alcohols and fatty
acids changed more gradually and reached a characteristic new pattern at d 57. In both compound classes
then, C30 compounds were slightly predominating.
The homolog distribution of alkanes was only
slightly altered in the same time interval, showing a
steady shift from compounds with ⱕ29 carbon atoms
to homologs with ⱖ31 carbons over time (data not
shown).
Because the homolog patterns of acetates, alcohols,
and fatty acids changed most drastically during leaf
development, the complete time course of these compounds has to be further inspected. To this end, the
amounts of even-numbered homologs centered
around C24 and C32, respectively, can be compared
because they showed most pronounced changes in
the investigated time interval (compare with Fig. 4).
Acetates with relatively short chain lengths were accumulated only before d 8 and then quickly lost (Fig.
5A). In contrast, higher acetate homologs were
slowly increasing during the first 23 d of development, then slightly decreasing and leveling off at
approximately 10 ␮g leaf⫺1. The latter time course
was exactly paralleled by both shorter and longer
homologs of alcohols (Fig. 5B). Only the fatty acids
with relatively short chain lengths showed a development similar to the corresponding alcohols,
whereas the longer fatty acid homologs were steadily
increasing over the whole time course (Fig. 5C).
The mechanical removal of superficial wax films
from immature leaves of P. laurocerasus was visualized by scanning electron microscopy (SEM). Two
days after bud break, the adaxial surface of leaves
was made up by undulated epidermal cells with
irregular polygonal outlines (Fig. 6A). Anticlinal cell
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Plant Physiol. Vol. 126, 2001
Leaf Epicuticular Wax Composition of Prunus laurocerasus
Figure 3. Development of epicuticular waxes
on adaxial surfaces of P. laurocerasus after an
initial treatment with gum arabic. A single treatment with gum arabic was employed to selectively remove surface waxes. Total amounts (average ⫾ SD [n ⫽ 6]; ␮g leaf⫺1) of epicuticular
waxes (gray area) and their composition were
determined by gas chromatographic (GC)
analysis.
boundaries were marked by sharp grooves, whereas
periclinal surfaces were structured by smooth ridges
and sporadic granules. Indentations delineating cell
boundaries were inversely replicated on the gum
arabic film removed from respective surfaces (Fig.
6B). Because substantial quantities of waxes were
detected in authentic gum arabic preparations, it can
be assumed that the polymer was coated with a wax
film. The visible surface thus represents the boundary layer between the mechanically removed waxes
and the remainder of the leaf cuticle. On very young
leaves, the treatment with gum arabic did not leave
microscopically visible traces. However, on leaves
more than 10 d old a rough line bordering the
polymer-treated area could be found (Fig. 6, C and
D). On the native surface of these leaves granular
structures had accumulated that were removed by
gum arabic together with the continuously underlying wax film. The largely unbroken film with interspersed granules alternatively could be deposited on
glass using cryo-adhesives as previously described
(Fig. 6, E and F). The film thickness increased markedly from approximately 25 nm at d 10 of the time
course to 100 nm at d 57.
In parallel to the developmental studies described
above, a selection of P. laurocerasus leaves was
probed with gum arabic without initial disruption of
the wax film (defining d 0 in the previous studies).
Leaves were sampled at developmental stages corresponding to d 5, 8, 10, and 57 in the original time
frame (see above). After removal of epicuticular
waxes respective leaves were harvested and adaxial
surfaces were extracted with chloroform. The resulting mixtures of intracuticular waxes were dominated
by the triterpenoids oleanolic and ursolic acid (Fig.
7). Their coverages increased drastically before d 8
and then slowly decreased from 900 to 600 ␮g leaf⫺1.
The aliphatic portions of the intracuticular mixtures
Plant Physiol. Vol. 126, 2001
were dominated by acetates at d 5, alcohols between
d 8 and 10, and alkanes thereafter. In the alcohol
fraction, homologs with chain lengths ⱕ C26 prevailed at all sampling points. In contrast, the acetates
consisted of relatively short chain lengths only at d 5,
whereas chain lengths ⱖ C28 predominated after d 8.
In a final experiment, the epicuticular wax film was
removed with frozen water from several developing
Figure 4. Chain length patterns of major aliphatic compound classes
in epicuticular waxes from adaxial P. laurocerasus leaf surfaces.
Homolog distributions (average ⫾ SD [n ⫽ 6]; %) in the acetate (A),
in the alcohol (B), and in the fatty acid fractions (C) were calculated
from individual compound yields in epicuticular wax mixtures sampled with gum arabic.
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Jetter and Schäffer
Figure 5. Time course of selected homologs of major compound
classes in epicuticular waxes from adaxial P. laurocerasus leaf surfaces. Coverages (average ⫾ SD [n ⫽ 6]; ␮g leaf⫺1) of individual
homologs were determined in epicuticular wax mixtures prepared
with gum arabic. Added amounts for acetate (A), alcohol (B), and
fatty acid homologs (C) with relatively short- (or long-) chain lengths
are plotted as a function of leaf development.
leaves (d 3 in the investigated time frame, corresponding to d 6 after bud break) and transferred to
glass supports. Individual preparations were stored
for 15 d directly at the mother plant (outdoors),
whereas others were kept in the greenhouse. After
this time, their composition was determined and
compared with that of the initial mixture and the
epicuticular wax film that meanwhile had developed
on the leaf surface. In vitro, both the relative composition and the coverage of all aliphatic film constituents, quantified as compound classes and as homolog
patterns therein, were unaltered (Fig. 8), whereas the
in vivo control confirmed the drastic decrease of
acetates and the accumulation of alcohols (see
above).
DISCUSSION
For the developmental investigations of the P. laurocerasus leaf surface, a method had to be devised
that would allow the nondestructive removal and
chemical analysis of waxes from living plant organs.
For this purpose, an adhesive film should be formed
on the leaf surface and then be removed together
1730
with adhering waxes without disturbing the tissue.
The adhesives that had previously been employed in
similar studies either could not sufficiently discriminate between intracuticular and epicuticular material (Haas and Rentschler, 1984) or included freezethaw cycles that impeded an in vivo application
(Jetter et al., 2000). We now found that both problems
could be overcome using gum arabic as an adhesive.
Parallel samples that were prepared from comparable P. laurocerasus leaves showed that coverages (␮g
cm⫺2) of individual compounds could be determined
with high reproducibility.
It had been reported that wax removal with cryoadhesives is highly selective for epicuticular material
because it relies entirely on the mechanical interaction between adhesive and wax films (Jetter et al.,
2000). For authentical material, the wax mixtures prepared with gum arabic and cryo-adhesives were
nearly identical in total amounts and composition.
Therefore, both methods show equally high selectivity for epicuticular waxes. This was further verified
by experiments in which the same leaf surface was
subjected to multiple gum arabic treatments and a
final extraction. The sharply declining yields of repeated mechanical wax removal showed that a physically resistant boundary had been reached. Beyond
this interface, more material could only be released
by solvent extraction. As for the cryo-adhesive technique, the physical boundary thus characterized
must be identical with the outer limits of the cutin
matrix. Hence, gum arabic treatment yielded exclusively epicuticular material, as defined by the location on the outside of the cutin matrix, whereas the
consecutive extraction released the remaining intracuticular material. The triterpenoid acids serve as
intracuticular markers being released neither by
cryo-adhesives nor by gum arabic treatment. Previous attempts to selectively prepare epicuticular
waxes by differential extraction (Silva Fernandes et
al., 1964; Holloway, 1974; Baker and Procopiou, 1975;
Baker et al., 1975; Svenningsson, 1988; Garrec et al.,
1995) suffered from cross-contamination with intracuticular waxes and could hence only qualitatively
describe surface compositions. With the new adhesive methods, the outermost layers of plant cuticles
now can be quantitatively analyzed.
In the course of the developmental investigation of
adaxial P. laurocerasus leaf surfaces, the mechanically
removed epicuticular wax films were visualized by
SEM. As judged after transfer to glass surfaces, the
epicuticular films on leaves of all developmental
stages were contiguous. After leaf expansion had
ceased, the film thickness increased steadily, corresponding to the observed increases in epicuticular
wax loads. For wax coverages of 3 and 6 ␮g cm⫺2
(corresponding to d 10 and 57 in the investigated time
frame), and based on density values of very-longchain aliphatic compounds of 0.8 to 1.0 106 g m⫺3 (Le
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Plant Physiol. Vol. 126, 2001
Leaf Epicuticular Wax Composition of Prunus laurocerasus
Figure 6. Scanning electron micrographs of adaxial P. laurocerasus leaf surfaces. A, Untreated control 2 d after bud break.
B, Gum arabic replica of the same developmental phase viewed from the side of the polymer that had been in contact with
the leaf. C and D, Surface of leaves 15 d after bud break: Granular structures mark the native surface (upper half) whereas
areas treated with gum arabic are smooth (lower half). Both zones are delineated by the rim of the epicuticular film (white
arrows). E and F, Film of epicuticular waxes from leaves 15 d after bud break (right side) transferred onto glass (left side) using
frozen water.
Roux, 1969), a film thickness of 30 to 40 nm and 60 to
75 nm can be expected, respectively. These chemical
estimates and the SEM representation of the epicuticular film thickness at various developmental stages
were hence in good accordance. For fully developed P.
laurocerasus leaves (more than 1 year old), an adaxial
wax film thickness of 130 to 160 nm had been reported
(Jetter et al., 2000) and the present data document a
steady net accumulation of material slowly forming
Plant Physiol. Vol. 126, 2001
the mature surface. Assuming that wax molecules are
all cis-configured (Small, 1984) and oriented perpendicular to the leaf surface (Sitte and Rennier, 1963) the
epicuticular film on 10-d-old leaves was approximately 10 molecules thick. During the following 50 d
its thickness had increased to approximately 20 molecular layers, whereas mature leaves showed a film
thickness corresponding to approximately 35 to 45
layers (Jetter et al., 2000).
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1731
Jetter and Schäffer
Figure 7. Composition of intracuticular waxes from adaxial sides of
P. laurocerasus leaves. Coverages (average [n ⫽ 2]; ␮g leaf⫺1) and,
for alcohols and alkyl acetates, relative portions of homologs ⱕC26
(lower part of bars) and homologs ⱖC28 (upper part of bars) are given.
The epicuticular wax film was removed initially to
create a controlled starting situation. Original wax
amounts and compositions were restored after approximately 3 d and further development of epicuticular waxes was not disturbed by the initial removal, as control experiments with single probing
(starting at d 5 in the present time frame) showed.
This demonstrates that epicuticular waxes on leaves
of P. laurocerasus can be regenerated at least in the
early stages of leaf development. In accordance with
this result, surfaces of diverse Eucalyptus and Brassica
species had previously been shown to reform epicuticular waxes especially on young leaves (Hallam,
1970; Percy and Baker, 1987; Wolter et al., 1988;
Wirthensohn and Sedgley, 1996). As in these studies,
the regenerated waxes were only monitored by SEM
and/or the originally removed wax layer was not
analyzed, the rates of wax restoration could not be
quantified. The regenerative capacities of plant surfaces can now be quantitatively studied as a function
of developmental stages. The epicuticular plasticity
of P. laurocerasus and other model species is currently
being investigated with the methods developed here.
Earlier reports on epicuticular waxes of mature P.
laurocerasus leaves were confirmed in the present
investigation, demonstrating that both the absolute
amounts and the relative composition of surface
compounds are invariable after leaf development is
completed (Stammitti et al., 1996; Jetter et al., 2000).
In contrast, drastic changes occurred during organogenesis: Various compound classes, most prominently acetates and alcohols, sequentially appeared
and were accumulated to high percentages at the
plant surface. It is of special interest that multiple,
distinct phases with extensive wax turnover occurred
within few days. Only moderate shifts, either in the
portions of compound classes, their coverages, or
their chain length distributions, had been reported
for waxes on other plant species (Tulloch, 1973;
Stocker and Wanner, 1975; Baker et al., 1982; Prasad
and Gülz, 1990; Gülz et al., 1991; Maier and Post1732
Beittenmiller, 1998; Rhee et al., 1998). These reports
could not specify where respective alterations occurred within the cuticle because mixtures of epiand intracuticular waxes had been analyzed. The
present findings confirmed the general trend for
longer chain homologs to accumulate in later developmental stages (Haas, 1977; Atkin and Hamilton,
1982; Jenks et al., 1996).
This is the first report on changes locally occurring
in the epicuticular wax film. As a consequence, the
question arises whether distinctive phases in epicuticular wax development are due to an ontogenetic
program or merely reflect differences in the flux of
biosynthetic products to the surface. First, intracuticular waxes contained high levels of triterpenoids
throughout leaf development. The newly appearing
aliphatic compounds hence had to move to the surface across a preformed intracuticular compartment.
Therefore, it can be excluded that the distinct epiand intracuticular regions were generated by sequential deposition of contiguous layers. Second, the
steady accumulation of aliphatics in the epicuticular
film, as monitored by changes in the amounts of
compound classes and chain length distributions,
was in all cases preceded by respective changes in the
intracuticular compartment. This is further evidence
for the separate movement of individual compounds
toward the surface. In conclusion, phase separation
likely occurs within the bulk cuticular waxes and
holds triterpenoids in the intracuticular compartment, whereas aliphatics are partitioned between
both layers.
Individual compound classes and homologs accumulate in the epicuticular compartment of P. laurocerasus leaves within a few days. Biosynthetic products must consequently move toward the plant
surface with rates in the order of nanograms per
centimeter2 an hour, in accordance with reports for
other plant species (Baker and Hunt, 1981). If both
phase separation and transport (within the cuticle)
Figure 8. In vitro and in vivo development of epicuticular wax films
from adaxial sides of P. laurocerasus leaves starting 5 d after budbreak. Wax films were transferred onto glass and stored for 15 d.
Coverages (average ⫾ SD [n ⫽ 5]; ␮g cm⫺2) of individual compound
classes were then compared both with the initial composition and to
the epicuticular film that had developed on control leaves.
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Plant Physiol. Vol. 126, 2001
Leaf Epicuticular Wax Composition of Prunus laurocerasus
are spontaneous then the resulting fluxes should be
limited by diffusion rates. According to Schreiber
and Schönherr (1993), the mobility of aliphatic molecules within the wax mixture will depend largely on
their chain length, but not on their functional groups.
Acetate and alcohol homologs centered around C24
should have diffusion coefficients of approximately
10⫺20 m2 s⫺1. Model calculations show that even with
this small molecular mobility an adequate flux of
material will spontaneously arise, if the compounds
are delivered to the innermost parts of the cuticle in
micromolar concentrations (Riederer and Schreiber,
1995). Because these concentrations are within reasonable limits, the flux rates of aliphatics to the leaf
surface of P. laurocerasus can be sufficiently explained
by diffusion.
The sequential appearance of diverse aliphatic
compound classes at the plant surface conversely
cannot be rationalized with differences in their diffusion rates. Based on their largely matching chain
length distributions these compounds, e.g. the acetates and alcohols in respective phases of leaf development, would be expected to diffuse with similar
rates (Schreiber and Schönherr, 1993). Instead of differential diffusion, the timed accumulation of epicuticular constituents therefore probably reflects the
ontogenetic regulation of wax biosynthesis (or apoplastic export). Therefore, it will be interesting to
compare future results on either the formation or the
export of wax constituents with the present details on
the temporal and spatial distribution of P. laurocerasus waxes.
Generally accepted models for the formation and
structure of the plant cuticle were assuming that
cuticular waxes are coordinately accumulated (Baker,
1982). In accordance, values for relative amounts (%)
or coverages (␮g cm⫺2) of individual constituents
may decrease over time when other compounds are
added or when leaves expand, respectively. However, losses of individual homologs or whole compound classes on a whole-leaf basis (␮g leaf⫺1) had
been reported only scarcely (Markstädter, 1994; Riederer and Markstädter, 1996; Hauke and Schreiber,
1998). In P. laurocerasus epicuticular waxes, both acetates and alcohols were found to decrease independently by almost 4 ␮g leaf⫺1 d⫺1 during special
periods in leaf development. Three alternative mechanisms could explain this loss of surface material: (a)
The compounds could be converted in situ into other
wax constituents, (b) they could be lost to the atmosphere by evaporation or erosion, or (c) they could be
transported back to inner parts of the cuticle, the
epidermal cell wall, or the protoplast, where they
would be metabolized or transformed into other wax
compounds.
The marked decrease of acetates in P. laurocerasus
epicuticular wax coincides with an increase of alcohols with identical chain lengths. Therefore, the latter
might be considered as in situ conversion products
Plant Physiol. Vol. 126, 2001
formed by acetate hydrolysis. This hypothesis was
tested in an experiment using the methods developed
here: Epicuticular wax films were isolated and exposed to natural climatic conditions. In vitro the
aliphatic film constituents were unaltered, whereas
the in vivo control confirmed the drastic changes
detailed above. Because the wax composition is not
affected when only the epicuticular film is exposed to
its natural atmospheric environment, a spontaneous
conversion of acetates into alcohols within the epicuticular waxes can be ruled out. This experiment also
shows that neither evaporation nor erosion can account for the loss of acetates from the epicuticular
film. Instead, the intracuticular compartment and/or
the epidermis must be involved in the processes.
Present evidence consequently favors the third mechanism, transport of epicuticular compounds back
into the tissue followed by further conversion, to
explain the loss of epicuticular compound classes
during P. laurocerasus wax development. Labeling
studies can be employed to further study the transport phenomena described in the present paper. With
the techniques developed here, the leaf surface can be
manipulated to perform adequate pulse-chase
experiments.
Coverages of the intracuticular triterpenoids of P.
laurocerasus leaves were diminishing with even
higher rates than epicuticular compounds. Similar
effects had previously been reported for triterpenoid
acids in leaf waxes of other Rosaceae, namely Malus
domestica (Hellmann and Stösser, 1992), Malus hupehensis, and Prunus cerasus (Baker and Hunt, 1981) as
well as Prunus persica (Baker et al., 1979). Although
epi- and intracuticular waxes were not differentiated
in these studies, it seems likely that, similar to the
closely related species P. laurocerasus, the triterpenoids are localized in inner parts of the cuticle. It has
been surmised that the yields of superficial solvent
extraction might not reflect the loss of compounds
but instead the changes in their intracuticular environment (Jetter et al., 2000). The slowly progressing
polymerization of the cutin matrix (Croteau and Kolattukudy, 1974; Schmidt and Schönherr, 1982; Kolattukudy, 1984; Khan and Marron, 1988; Riederer and
Schönherr, 1988) could gradually restrict access for
solvent molecules and would hamper extraction. The
triterpenoid acids alternatively could react with functional groups on the cutin matrix, binding them to
the polymer and hence rendering them insoluble.
Similar processes have been reported for aromatic
acids (Basford, 1991) and fatty acids (Hauke and
Schreiber, 1998). Although these mechanisms could
explain the reduced yields of intracuticular triterpenoid acids from older leaves, they cannot account for
the losses of epicuticular acetates and alcohols.
Hence, two different mechanisms, translocation of
epicuticular waxes and changes in the intracuticular
structure, might lead to the apparent loss of compounds from cuticular compartments.
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
1733
Jetter and Schäffer
The present data set bears implications related not
only to cuticle structure and formation but also to the
ecological and physiological properties of plant surfaces. The developmental changes in P. laurocerasus
leaf wax composition require a precise regulation of
wax biosynthesis and an investment of energy and
material. Therefore, it seems very likely that specific
functions are correlated with the different compound
classes dominating the surface composition in distinct developmental phases. Because the accumulation of acetates coincides with the period of fastest
leaf expansion, this compound class might play a
special role in sealing the still thin, rapidly expanding cuticle (Schönherr, 1976; Schönherr and Riederer,
1988). On the other hand, the transient amassing of
alcohols in the epicuticular film brings about that the
surface composition of the developing leaf completely changes twice in short intervals. This might
help to divert herbivorous insects that use either wax
acetates or alcohols or alkanes as host surface markers by shortening the time windows in which the
animals can recognize their host (Städler, 1986;
Carver et al., 1990; van Loon et al., 1992; Podila et al.,
1993; Flaishman et al., 1995). Our new methods allow
to analyze and manipulate surface composition in
situ. Hence, both the physiological and the ecological
properties of the epicuticular wax film at different
developmental stages can now be investigated.
MATERIALS AND METHODS
Plant Materials
Small trees of Prunus laurocerasus were continuously
grown in pots in the greenhouses of the Botanical Garden
of the University of Würzburg. For method development,
mature leaves were harvested randomly from three individual plants in fall and winter and were pooled for wax
removal and analysis. Leaf discs with 20-mm diameter
were cut from these leaves and used to compare the reproducibility and selectivity of mechanical wax removal using
either cryo-adhesives or gum arabic.
A single tree was moved into the garden in the springtime 2 weeks before developmental studies of epicuticular
waxes began. All available leaf buds on this plant were
marked and inspected regularly. Batches of 18, 14, 19, 24,
and 15 fresh leaves on various twigs were selected that had
bud breaks on April 25, April 28, May 2, May 11, and May
15, respectively. Three days after their individual bud
break, leaves were treated once with gum arabic (see below) to remove the epicuticular waxes from the entire
adaxial surface, thus defining d 0 in the time course. After
a defined number of days the same leaf surfaces were
treated a second time with gum arabic to sample epicuticular waxes for quantitative and qualitative chemical analyses. Individual leaves were sampled and analyzed separately. At each sampling time the epicuticular wax film on
the entire adaxial surface was probed for six individual
leaves in parallel. Wax development and leaf growth were
1734
monitored for 85 d after bud break. Data are shown for the
first 57 d of development.
Surface areas of all the investigated leaves had to be
monitored individually at respective sampling days without disturbing the leaf or touching the adaxial surface.
Because the overall shape of leaves did not change markedly during development, digital representations of model
leaf outlines could be interpolated by superposition of
multiple leaves of varying age. Only the length and the
width of the leaves used for wax sampling then were
measured and used to reconstruct computer images of the
leaves and calculate their surface areas. Based on these
calculated leaf areas, wax coverages per leaf were finally
determined for all sampling times. To corroborate the calculation results, surface areas of all investigated leaves
were additionally determined gravimetrically after the last
wax sampling using photocopies of the leaves. The surface
areas of all individual leaves determined with this method
were 17% ⫾ 4% larger than the calculated values used in
the present investigation.
Small pieces (0.1–0.5 cm2) of the gum arabic films removed from the adaxial surfaces of leaves at various developmental stages (see below) were used to assess epidermal cell densities. The partially translucent polymer films
were viewed at 20-fold magnification under a light microscope (Zeiss Axioplan, Oberkochen, Germany). Cuticular
ridges along the anticlinal walls of epidermal cells gave
dark contrasts indicating the cell outlines. These contours
were used to count epidermal cells within predefined areas
(0.2 ⫻ 0.2 mm) and calculate cell densities. In parallel,
Collodion replicas of adaxial surfaces of other leaves were
prepared at various developmental stages. Their investigation yielded cell densities identical to those determined
with gum arabic. With both methods only the central part
of the leaf surface was probed to minimize fluctuation that
could be caused by differential termination of cell division
and expansion in various parts of the leaf.
Mechanical Wax Removal
A polymer film of gum arabic was employed for the
selective preparation and analysis of epicuticular waxes.
Commercial gum arabic (Roth, Karlsruhe, Germany) was
extracted exhaustively with hot chloroform to remove any
soluble lipids and residual organic solvent was allowed to
evaporate completely. Approximately 0.1 mL of a 90%
(w/w) aqueous solution of pretreated gum were applied
per centimeter2 of leaf surface using a small paintbrush.
After 1 to 2 h, a dry and stable polymer film had formed
that could be broken off in pieces. These were collected and
transferred into a vial containing 7 mL each of chloroform
and water. The polymer films either from five 3.1-cm2 discs
(cut from mature leaves) or from entire adaxial surfaces of
intact individual leaves were pooled into the same twophase system. Tetracosane was added as internal standard,
after vigorous agitation and phase separation the organic
solution was removed and the solvent was evaporated
under reduced pressure.
Alternatively, either frozen glycerol or water was employed for the selective removal and analysis of epicuticu-
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 126, 2001
Leaf Epicuticular Wax Composition of Prunus laurocerasus
lar waxes as described elsewhere (Jetter et al., 2000). After
treatment with either glycerol or gum arabic, the leaf discs
and leaves were still physically intact and could be used in
repeated mechanical removal experiments or for superficial solvent extraction of the remaining cuticular waxes.
Wax Extraction
Selective superficial extraction of cuticular waxes from
the adaxial surface was achieved by placing the intact leaf
onto a flexible rubber mat, gently pressing a glass cylinder
with 10-mm diameter onto the exposed surface, and filling
the cylinder with approximately 1.5 mL of chloroform. The
solvent was agitated for 30 s by pumping with a Pasteur
pipette and removed. This procedure was repeated once
and both extracts were combined. When any solvent leaked
between cylinder and leaf surface the sample was discarded. Extracts from five individual leaves were pooled
for further analysis. Tetracosane was immediately added to
all the extracts of cuticular waxes as internal standard and
the solvent was removed under reduced pressure.
Chemical Analysis
Prior to GC analysis, hydroxyl-containing compounds in
all samples were transformed to the corresponding trimethylsilyl derivatives by reaction with bis-N,O-trimethylsilyltrifluoroacetamide (Macherey-Nagel, Düren, Germany) in
pyridine (30 min at 70°C). The composition of the mixtures
was studied by capillary GC (5890 II, Hewlett-Packard,
Avondale, PA) with on-column injection (Hewlett-Packard
30-m OV-1 WCOT i.d. 320 ␮m, Chrompack, Middelburg,
The Netherlands) and mass spectrometric detector (70 eV,
m/z 50–650, and Hewlett-Packard 5971). GC was carried out
with temperature programmed injection at 50°C, oven 2 min
at 50°C, 40°C min⫺1 to 200°C, 2 min at 200°C, 3°C min⫺1 to
320°C, 30 min at 320°C, and He carrier gas inlet pressures
programmed 5 min at 50 kPa, 3 kPa min⫺1 to 150 kPa, and
30 min at 150 kPa. Wax components were identified by
comparison of their mass spectra with those of authentic
standards and literature data. For quantification of individual compounds, GC was used under conditions as described
above, but with carrier gas H2 (5 min at 5 kPa, 3 kPa min⫺1
to 50 kPa, and 30 min at 50 kPa) and flame-ionization
detector.
SEM
Epicuticular wax films were removed from the adaxial
surface of P. laurocerasus leaves employing either frozen
water as a cryo-adhesive (Jetter et al., 2000) or gum arabic
as described above. Cryo-adhesive preparations were
placed bottom-down on glass slides and air dried. This
procedure exposed the original outer surface for inspection
by SEM. Gum arabic treatments alternatively were placed
bottom-up on glass slides, thus allowing to view the original inner surface of the epicuticular wax film. The treated
leaves and the glass slides carrying the wax preparations
were mounted on aluminum holders, sputtered with 20 nm
of gold (Balzers Union Sputtering Device, 25 mA, 300 s),
and investigated by SEM (Zeiss DSM 962, 15 kV, 6 mm).
Plant Physiol. Vol. 126, 2001
ACKNOWLEDGMENTS
Technical assistance by the Department for Electron Microscopy and by the Botanical Garden of the University of
Würzburg is gratefully acknowledged. We would also like
to thank Prof. Dr. Markus Riederer (University of Würzburg) and Dr. Chris E. Jeffree (University of Edinburgh,
UK) for numerous fruitful discussions.
Received February 20, 2001; returned for revision April 6,
2001; accepted May 21, 2001.
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