Journal of Experimental Botany, Vol. 50, No. 331, pp. 175–182, February 1999
Structure and dynamics of reconstituted cuticular waxes
of grape berry cuticle (Vitis vinifera L.)
Carolina G. Casado and Antonio Heredia1
Departamento de Bioquı́mica, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, España
Received 20 June 1998; Accepted 11 September 1998
Abstract
Cuticular waxes from grape berry cuticle of Vitis vinifera L. have been investigated by X-ray diffraction,
differential scanning calorimetry and gas chromatography. The waxes were mainly composed of n-alcohols and n-fatty acids and a considerable amount
(about 30%) of the cyclic terpenoid oleanolic acid. The
physical techniques used showed that the waxes have
a high degree of molecular order in spite of the presence of the cyclic component. Molecular dynamics of
the reconstituted waxes were measured to characterize the transport properties of the cuticular waxes that
form the transport-limiting barrier of plant cuticles.
For this purpose, the diffusion coefficient of labelled
cholesterol, imitating the terpenoic acid, was measured. The value obtained was around 10−21 m2 s−1
indicating a low mobility of the cyclic part of the reconstituted waxes. Temperature dependence of the diffusion coefficient was studied in the range of 5–45 °C.
Arrhenius plot analysis yielded a high activation
energy, 196.4 kJ mol−1, of the diffusion process. This
indicates dense molecular packing of reconstituted
cuticular waxes.
Key words: Waxes, structure, grape cuticle, oleanolic acid,
Vitis vinifera.
Introduction
The leaf and fruit surface of higher plants are covered by
the cuticle or cuticular membrane. Thus, the plant cuticle
constitutes the interface between the plant tissue and the
environment. From a structural point of view, the cuticle
is formed by an insoluble polymer matrix of hydroxyfatty acid esters called cutin. Associated with this biopolymer are waxes or soluble cuticular lipids; embedded
within the matrix, intracuticular waxes or deposited on
the outer surface of the plant cuticle, epicuticular waxes.
Cuticular waxes constitute the main barrier limiting the
transport across the plant–atmosphere interface. This property allows waxes to control cuticular transpiration
(Schönherr, 1982), foliar uptake of xenobiotics (Schönherr
and Riederer, 1989; Schreiber and Schönherr, 1993) and
resistance against fungi (Comménil et al., 1997). This is due
to their partial arrangement in crystalline regions, where
the middle portions of the long aliphatic chains of the wax
constituents are regularly aligned, and in amorphous
regions where short-chain aliphatics and cyclic compounds
form clusters outside crystalline regions (Riederer and
Schreiber, 1995). Those waxes can, in fact, form crystalline
platelets oriented parallel to the surface of the cuticular
membrane and these have been demonstrated using polarized light (Sitte and Rennier, 1963).
Permeability of cuticles differs greatly among plant
species and it has been argued that both the chemistry
and the structure of cuticular waxes are responsible for
these differences (Riederer and Schönherr, 1985). Most
studies have been carried out in plant species where
epicuticular waxes are mainly composed of long aliphatic
molecular chains. In this sense, current knowledge of the
fine structure and dynamics of different plant cuticular
waxes is limited (Riederer and Schreiber, 1995).
The epicuticular waxes of grape berry fruit constitute
about 25% of their cuticular membrane weight. They are
mainly formed by oleanolic acid, a cyclic triterpenoid
acid, which can constitute up to 60% of the total wax
(Comménil et al., 1997). In minor and variable proportions, a mixture of fatty acids, esters, primary alcohols,
and other aliphatic compounds are also present (Radler
and Horn, 1965; Comménil et al., 1997). Thus, these
waxes could be a good model to complete our understanding of plant waxes.
The present work reports on the physical and chemical
1 To whom correspondence should be addressed. Fax: +34 52 132000. E-mail: [email protected]
© Oxford University Press 1999
176
Casado and Heredia
characterization of epicuticular grape fruit waxes in order
to elucidate the exact role of non-aliphatic wax components in the fine structure and molecular arrangement of
this wax model.
Materials and methods
Plant material
Bunches of grapes were collected from Vitis vinifera L. cv.
Palomino fino from Domecq vineyards located at Jerez de la
Frontera, Cádiz, Spain. The material was collected at the end
of July, when it was beginning its maturity stage.
Isolation of epicuticular waxes
Fresh plant material was washed in water, dried on filter paper,
and cuticular waxes were extracted by immersing the berries in
chloroform for 60 s. The extract was filtered after mixing the
organic solvent with solid anhydrous sodium sulphate. Finally,
the organic solvent was evaporated at room temperature and
the waxes were stored at room temperature until they were
used in the corresponding experiments.
Identification and quantification of cuticular waxes
The extracts of cuticular waxes were analysed by combined gas
chromatography-mass spectrometry (GC-MS) in a HewlettPackard 5890 ( USA) with a methyl silicon capillary column. For
derivatization, the samples were dissolved in 100 ml of N,Obis(trimethylsilyl )acetamide (Sigma) and 100 ml of dry pyridine
(Aldrich) for 30 min at 60 °C (Soliday et al., 1979). Peak areas
were recorded by electronic integration and the specific correction
factor was applied to the peak area of oleanolic acid. All
dominating compounds were identified and they comprised more
than 85% of the chromatogram area. Single components were
identified by comparing their mass spectra with those from
autenthic samples (oleanolic acid), from the NBS library of mass
spectra and from previously published data.
X-ray diffraction
X-ray diffraction patterns were obtained with a Siemens D-501
automated diffractometer (Siemens, Germany) using graphitemonochromated CuK radiation. The diffractograms were
a
generally recorded between 4° and 40° (2h) in 0.04° steps,
counting 4 s per point, at 40 kV and at 20 mA, where 2h is the
angle between the direction in which the scattered amplitude
after diffraction is measured and the direction of the incident
radiation. The samples were placed on a glass support.
Measurement of diffusion coefficient
In order to measure the diffusion coefficient, the experimental
approach described by Schreiber and Schönherr (1993) was
used: isolated cuticular waxes or in some experiments, pure
oleanolic acid (Aldrich) or hexacosanol (Aldrich) dissolved in
chloroform were mixed with 3H-labelled cholesterol and spread
on aluminium discs (about 13 mm diameter). The 3H-labelled
cholesterol had a specific activity of 1.74 TBq mmol−1
(Amersham, England ). The mass ratio of the amount of added
radioactive compound to the amount of wax sample was always
about 1:1000. After evaporation of the solvent, the aluminium
discs were heated, in order to obtain good adhesion of the wax
to the metal surface, followed by cooling to room temperature.
Desorption experiments were carried out using a soybean
lecithin phospholipid suspension (PLS ) (1% in water; Sigma).
Desorption kinetics were measured using 7.5 ml glass vials
containing 7 ml PLS and one single wax sample. The vials were
kept on a shaker bath and rotated horizontally, 80 rpm at
25 °C. At known time intervals the desorption medium was
quantitatively exchanged and replaced by fresh PLS. At the
end of the desorption experiments, wax samples were collected
and the residual amount of radioactivity in the wax was
determined by dissolving the wax in chloroform. Radioactivity
of the PLS and organic solutions were determined in special
liquid scintillation cocktails (Aquasol-2 NEF-952G, Belgium;
BCS-NA, Amersham, England, respectively).
Desorption kinetics are described using the equation
(Schreiber and Schönherr, 1993):
M /M =(4/Dx)(D/p)1/2t1/2
(1)
t 0
where M /M is the relative amount of radioactivity desorbed,
t 0
Dx (m) is the thickness of the wax layer, D (m2 s−1) is the
diffusion coefficient and t (s) is the time. Plotting M /M against
t 0
the square root of time the desorption curves could be linearized
up to about 50% desorption. The diffusion coefficient can be
calculated from the slopes of the corresponding regression lines,
fitted to the linear parts of the desorption kinetics, since they
correspond to the term (4/Dx)(D/p)1/2 of equation (1).
Calorimetry
All reported experiments were performed in a Shimadzu DSC-50
differential scanning calorimeter (DSC, Shimadzu Corporation,
Japan) with computer-aided data analysis. All experiments
followed the same protocol (Luque and Heredia, 1997).
Aluminium DSC pans containing between 1.2 and 2.2 mg per
sample, were loaded into the calorimeter at room temperature.
For each run, the thermograms were recorded during heating
at 5 °C min−1 or 10 °C min−1 and post-cooling at 5 °C min−1
under a flow of nitrogen gas of 10 ml min−1. The scanning rate
elected gave good reproducibility and accuracy in the temperature and energy determinations. Second-order transitions were
detected by a shift in power of the baseline. Thermograms
recorded from different samples gave practically the same
transition temperatures. To obtain the thermogram of a sample,
the baseline was subtracted from the data obtained from
each sample.
Molecular modelling
Theoretical calculations were made using the HyperChem
software package (Hypercube, Waterloo, Canada). Molecular
mechanics calculations were carried out using default parameters
and standard parametrization using the Newton–Raphson
algorithm.
Results and discussion
Composition of grape berry cuticular waxes
Analysis of the cuticular wax isolated from mature grape
berry by combined GC-MS showed the presence of different
components (Table 1). The main component identified was
the triterpernoic acid, oleanoic acid, which constituted
nearly 30% of the total weight of the extract of cuticular
wax and, in different proportions, a mixture of long-chain
alcohols and fatty acids (Table 1). Other aliphatic compounds were found at lower amounts. This was the case of
n-alkanes and long chain esters. Oleanolic acid (C H O )
30 48 3
was identified from its mass spectral data of the correpond-
Physico-chemical characterization of cuticular waxes 177
Table 1. Quantitative composition of isolated grape berry waxes
Relative composition of the different fractions of cuticular wax from
mature grape berry cuticle (Vitis vinifera L.)
Compound
Percentage of mass (%)
Oleanolic acid
n-Fatty alcohols
C
22
C
24
C
26
C
28
n-Fatty acids
C
24
C
26
C
28
Others (n-alkanes and long chain esters)
29.7
1.3
12.0
23.5
12.1
7.8
7.6
1.0
5.1
ing trimethylsilyl derivative. These results are in good agreement with those obtained by Radler and Horn (1965) and,
more recently, by Comménil et al. (1997). These authors
reported that the cuticular waxes predominantly consisted
of fatty alcohols and oleanolic acid. Other aliphatic compounds, such as wax esters and n-alkanes or hydrocarbons,
became significant during the maturation of the fruit
(Comménil et al., 1997). In our case, since the grape berries
were collected at the beginning of the maturation, the wax
samples only contained small amounts of these aliphatic
wax components.
a first order transition, characterized by an endothermic
at 72 °C, and a wide second-order thermal transition
appearing at approximately 120 °C and extended up to
350 °C. The endothermic recorded at 72 °C can be attributed to the melting of aliphatic components of the wax
sample. It is noticeable that a synthetic mixture formed
by hexacosanol and tetracosanoic acid (ratio 151) gave
an endothermic at 75 °C (data not shown). On the other
hand, the wide endothermic recorded at higher temperatures could be assigned to the oleanolic acid, which has
a melting point of 310 °C (Merck Index, 1996). It is
important to emphasize that these results agree well with
the composition found in the cuticular waxes investigated
in this work describing the major components of the wax
samples investigated here: oleanolic acid and fatty acids
and alcohols. The presence of significant amounts of
other wax components such as n-alkanes or wax esters
are characterized by a thermogram with meltings around
65 °C (e.g. C n-alkane) or between 80–85 °C (C –C
31
42 48
long chain esters).
These results also suggest that the long aliphatic chains
of the fatty alcohols and acids of the wax investigated
here are regularly aligned, forming solid crystalline parts
characterized by sharp endothermic peaks which are
embedded within a matrix of less crystalline material,
characterized by a wider endothermic peak, formed
mainly by the triterpenoid oleanolic acid.
Calorimetric behaviour of cuticular wax
Structural information of wax samples can be obtained
by differential scanning calorimetry (Luque and Heredia,
1997). The thermogram recorded for the grape berry
cuticular waxes is shown in Fig. 1. The thermogram shows
X-ray diffraction analysis
The X-ray diffraction technique is a tool which allows
information to be obtained on the molecular arrangement
at the atomic level. In the solid state, this is by far the
Fig. 1. DSC heating thermogram, between 25 °C and 360 °C, of reconstituted cuticular waxes isolated from grape berry. The thermogram was
recorded under non-oxidative conditions.
178
Casado and Heredia
Fig. 2. X-ray diffraction patterns of isolated grape berry cuticle, reconstituted cuticular grape berry waxes and n-hexacosanol. Traces shifted to
each other for clarity.
Fig. 3. X-ray thermodiffraction pattern of reconstituted cuticular waxes from grape berry. Traces shifted to each other for clarity.
most powerful method for structure determination. It has
been succesfully used by some research groups on different
aspects of plant cuticular membranes (Reynhardt and
Riederer, 1991; Luque et al., 1995; Viougeas et al., 1995).
The X-ray diffraction pattern of reconstituted cuticular
waxes of grape berries is shown in Fig. 2. The X-ray
diffractogram shows diffraction peaks from lattice planes
of the crystalline wax zone only. The amorphous band, a
less ordered component of the wax, contributed only to
a broad band characteristic of diffraction from noncrystalline material, ranging from ~9° to 35° ( Fig. 2).
The two narrow peaks of high intensity, superimposed
on the broad peak, are the so-called 010 and 100
reflections and correspond to basal spacings at 4.17 and
3.74 Å, respectively. It is interesting that an isolated
cuticular membrane from grape berry showed an X-ray
Physico-chemical characterization of cuticular waxes 179
diffraction pattern which revealed the presence of the
crystalline structures (Fig. 2). These peaks appeared on
a very broad band of high intensity at ~21°, and were
mainly attributed to the amorphous network that constitutes the cutin of the plant cuticle (Luque et al., 1995).
The recorded sharp peaks mentioned above can be
clearly attributed to the mixture of fatty acids and alcohols identified in the wax sample ( Table 1), because the
corresponding X-ray pattern of pure hexacosanol (C
26
fatty alcohol ) showed two peaks of high intensity and
the same intensity ratio located at 4.10 and 3.65 Å,
respectively (Fig. 2). Since these wax components have
very long c axes, reflections from 00l planes were observed
at small angle diffraction peaks ( Fig. 2). It is important
to point out that normal aliphatic long-chain compounds
crystallize as piles of single molecular layers with the
carbon chains in parallel, in upright or tilted positions.
Their X-ray diffraction patterns usually show two sets of
diffraction peaks, one at a normal diffraction angle and
another at a small diffraction angle. The former depends
on the side spacings between the chains (the distances
measured at 4.10 and 3.65 Å) and the latter on the
thickness of the layers and, hence, on the length and
nature of the chains and their angle with the basal plane.
The fact that no such diffraction peaks were observed for
the reconstituted waxes recorded in Fig. 2, suggests that
due to either the wide distribution of the chain-lengths
or the different orientations of the crystals, the 00l planes
are not well-defined in the wax sample.
A supplementary and broader peak appeared at ~16°
( Fig. 2). The peak has low intensity and corresponds to
a basal spacing of 5.7 Å. This peak appears neither in the
X-ray diffraction pattern of any fatty acid or fatty alcohol
nor generally in the diffraction patterns of aliphatic
organic compounds of high chain length (Heyding et al.,
1990). This basal spacing can be assigned to the molecular
arrangement of the oleanolic acid of the wax sample. In
spite of this compound being the main component of
grape berry wax, its contributions to the crystallinity of
the reconstituted waxes is very low. This is an indication
of the low ordering of oleanolic acid in the wax sample
and, for these reasons, the triterpenoid acid can be
considered as the component of the solid amorphous-like
phase of the wax samples. Nevertheless, it is important
to emphasize the existence of a determined molecular
order attributed to the cyclic component of grape berry
cuticular wax. The wax model developed by Riederer and
Schreiber (1995) indicates that cyclic wax components
constitute the so-called solid amorphous and amorphous
phases of wax in contrast to the crystalline part mainly
formed by aliphatic components. At the molecular level,
this is the first research on the structural role of cyclic
wax components indicating the molecular arrangement
of this type of compound. From the data reported here,
it can be affirmed that oleanolic acid forms a phase wax
matrix with a very distinct molecular ordering to what is
usually called the amorphous phase.
A definitive probe on the assigment of the peak
recorded at 5.7 Å, in addition to more detailed information at the structural level, could be achieved by monitoring the diffraction peaks of a sample of reconstituted
waxes with temperature. Figure 3 shows the different
diffraction patterns of a wax sample during heating, from
room temperature to 100 °C. While the peaks located at
4.17 and 3.74 Å clearly disappear between 60 °C and
100 °C due to the melting of the crystalline mixture of
fatty acids and fatty alcohols, the diffraction peak of low
intensity recorded at ~16° and assigned to the arrangement of oleanolic acid remained unaffected by temperature. These data agree with the calorimetric behaviour
discussed above.
Dynamics of the reconstituted cuticular waxes
The analysis of molecular dynamics and transport properties of grape berry cuticular waxes was carried out using
Fig. 4. (A) Relative amount of 3H-cholesterol desorbed (M /M ) versus
t 0
time and (b) versus square root of time, measured with reconstituted
grape berry waxes. A regression line (b) was fitted to the linearized
desorption kinetics. Bars represent 95% confidence intervals.
180
Casado and Heredia
the experimental approach developed by Schreiber and
Schönherr (1993). This method permits the measurement
of the diffusion coefficient of radiolabelled organic components incorporated into reconstituted cuticular waxes.
The diffusion coefficient can be considered as a direct
measurement of the mobility of the chemical in the
cuticular wax solid matrix.
The grape berry cuticular waxes consist of a crystalline
zone containing mainly the tails of the long-chain alcohols
and acids and a significant and extended amorphous zone
or mobile amorphous zone in the terms of Reynhardt
and Riederer (1994). In order to measure the transport
properties of this particular type of isolated waxes, it was
considered interesting to use a mobility probe essentially
located in this less crystalline wax phase. 3H-cholesterol
was used because of its high structural and chemical
similarity to the triterpenoid oleanolic acid, the main nonaliphatic constituent of the wax samples.
Desorption of 3H-labelled cholesterol, the wax-like
molecular probe, from reconstituted grape berry wax with
inert PLS desorption medium resulted in non-linear
desorption kinetics as shown in Fig. 4a. Diffusion coefficients (D) were calculated from the linearized part of the
desorption kinetics ( Fig. 4b) according to equation
(1).3H-cholesterol diffusion coefficient calculated from
these figures was 3.0±1.15×10−21 m2 s−1. This value is
very low and it reaches the range of diffusion coefficients
reported by Schreiber et al. (1996) which covered values
between 10−22 and 10−17 m2 s−1. These data show the
extremely low mobility of the less crystalline or more
amorphous phase of grape berry epicuticular waxes. The
value obtained could be related to the molecular size of
the oleanolic acid: it has been reported that there is an
exponential dependence of the diffusion coefficients on
molar volumes, which allows cuticular transport properties to be related to the physical structure of the wax
(Schreiber et al., 1996). Nevertheless, the diffusion
coefficient obtained in this work is 15 times lower than
that measured by Schreiber et al. (1996) in reconstituted
cuticular waxes of Fagus sylvatica and Picea abies using
cholesterol as the labelled molecular probe. These data
indicate that the mobility of cholesterol represents in fact
the mobility of the less ordered phase of the cuticular
waxes studied in this work.
When the labelled cholesterol is mixed with pure
oleanolic acid and the corresponding desorption experiments
performed,
a
diffusion
coefficient
of
63.8±22.2×10−21 m2 s−1 was obtained. This value is
higher but comparable to the one measured in reconstituted grape berry cuticular waxes. On the other hand,
when the same experiment was conducted in a matrix of
pure and crystalline hexacosanol, the C fatty alcohol, a
26
diffusion coefficient of 1.27±0.05×10−18 m2 s−1 was
obtained. These data reflect the exclusion of the labelled
molecular probe from the crystalline fatty alcohol.
Fig. 5. Arrhenius representation obtained for diffusion of 3H-cholesterol
in reconstituted cuticular waxes from grape berry. Bars represent 95%
confidence intervals.
Fig. 6. Molecular model of a dimer of oleanolic acid obtained by
molecular mechanics. For details, see text.
In order to obtain more structural information on the
more amorphous wax components of grape berry cuticular waxes, the effect of temperature on molecular mobility of the molecular probe was studied. Thus, temperature
dependence of diffusion coefficients was quantified using
the Arrhenius equation:
D=D exp–(E /RT )
(2)
0
D
where D is the pre-exponential factor, E (kJ mol−1)
0
D
the activation energy of the diffusion process, R the gas
constant and T ( K ) the temperature. The corresponding
Arrhenius plot ( Fig. 5) was linear in the temperature
Physico-chemical characterization of cuticular waxes 181
range investigated (5–45 °C ) indicating that temperature
changes did not cause either phase transition or other
structural changes in the reconstituted waxes. This result
agrees well with the calorimetric behaviour discussed
above. It is clear that the mobility of cholesterol in the
reconstituted waxes investigated increased with temperature. Between 5 °C and 45 °C the measured diffusion
coefficient increased almost 100 times. The activation
energy calculated from the slope of the Arrhenius plot
( Fig. 5) was 196.4 kJ mol−1. This value is very similar to
one recently published by Baur et al. (1997) for the
diffusion of the same molecular probe in isolated Hedera
helix cuticles. On the other hand, the obtained activation
energy can be considered high when this value is compared
to the one calculated by Baur et al. (1997) for other
molecular probes in isolated fruit and leaf cuticles.
If it is considered that the diffusion coefficient of
cholesterol reflects the mobility of the less ordered phase
of the grape berry cuticular waxes, the elevated E
D
obtained implies dense packing and molecular arrangement in this wax phase since high energy is required to
produce holes or vacancies sufficiently large to accommodate the diffusing molecules (Baur et al., 1997).
The fact mentioned above can be illustrated after
examination of the molecular model shown in Fig. 6
which represents the optimized molecular geometry of a
dimer of oleanolic acid using molecular mechanics as
methodology. This theoretical dimer shows hydrogen
Fig. 7. Molecular model of the arrangement of the main components, oleanolic acid and n-hexacosanol, that constitute the cuticular wax of
grape berry.
182
Casado and Heredia
bond interactions between the hydroxyl group of one
molecule of the triterpenoic and the carboxylic acid group
of the second molecule. This conformation has two
important structural characteristics. First, the remaining
hydroxyl and carboxylic functional groups of both molecules could interact with complementary functional groups
of other molecules of oleanolic acid and following this,
conduce the formation of a tridimensional arrangement.
Secondly, the dimer conformation obtained by theoretical
molecular mechanics calculations is separated by an average distance of 6.2 Å, a value very close to the experimental basal spacing assigned to the oleanolic acid after
X-ray diffraction.
From molecular mechanics calculations, a putative
arrangement of cuticular grape berry wax is illustrated in
Fig. 7. Together with crystalline planes, mainly consisting
of the C alcohol, a less ordered matrix of oleanolic acid
26
dimers appears in very different orientations.
Finally, the authors would like to emphasize the consequences of the extraordinarily developed molecular
barrier that constitutes the cuticular wax of grape berry
cuticles. From a physiological point of view, the high
degree of order observed at the molecular level represents
a formidable barrier to protect and give consistency to
the grain of grape berries. These structural characteristics
could have physiological importance against water loss
from the fruit and fungus infection. Some of these properties will need further research.
Acknowledgements
The authors thank Mr Antonio Matas for his technical
assistance in the molecular modelling calculations. Financial
source: DGICYT (project PB94–1492) and Plan Nacional I+D
(project PTR94–0068).
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