Annals of Botany 87: 641±648, 2001
doi:10.1006/anbo.2001.1387, available online at http://www.idealibrary.com on
Analysis of Epicuticular Phenolics of Prunus persica and Olea europaea Leaves:
Evidence for the Chemical Origin of the UV-induced Blue Fluorescence of Stomata
G E O R G I O S L I A KO PO U LO S , S OT I R I A S TAV R I A N A KO U
and G E O R G E K A R A B O U R N I OT I S *
Laboratory of Plant Physiology, Department of Agricultural Biotechnology, Agricultural University of Athens,
Iera Odos 75, 11855 Botanikos, Athens, Greece
Received: 4 October 2000 Returned for revision: 28 December 2000
Accepted: 22 January 2001 Published electronically: 26 March 2001
Epicuticular waxes were removed from the leaf surfaces of Olea europaea and Prunus persica by washing with
chloroform and the resulting rinses were analysed by high performance liquid chromatography (HPLC) for the
presence of ¯uorescing compounds. Removal of epicuticular waxes from leaves of some representative plants by the
same treatment resulted in a signi®cant reduction in the intensity of the blue ¯uorescence emitted from guard cells
(Karabourniotis et al., 2001: Annals of Botany 87: 631±639). Ferulic acid and p-coumaric acid, as well as a number of
unidenti®ed compounds, were constituents of the rinses of both plants examined but only after alkaline hydrolysis of
the samples. This indicates that both phenolic acids are tightly bound to the epicuticular waxes of the leaves of these
plants. HPLC chromatograms of rinses derived either from the abaxial or adaxial surfaces of the hypostomatic leaves
of O. europaea did not show signi®cant qualitative di€erences. Nevertheless, ferulic acid (the main blue ¯uorescent
component) was much more abundant in the abaxial than the adaxial surface. In P. persica, the composition of the
sample derived from the abaxial surface was far more complex, and all constituents were present in much higher
concentrations than in the sample derived from the adaxial surface. Given the particular ¯uorescence characteristics
of ferulic acid, the di€erences in its concentration between abaxial and adaxial surfaces, and between the two species,
and the ¯uorescence images of these surfaces under the microscope, we propose that this compound is probably the
main epicuticular constituent responsible for the blue ¯uorescence emitted by guard cells of the species examined. The
# 2001 Annals of Botany Company
functional signi®cance of the ®ndings is discussed.
Key words: Cuticle, epicuticular waxes, ferulic acid, HPLC analysis, Olea europaea L., p-coumaric acid, phenolics,
Prunus persica L., stomata.
I N T RO D U C T I O N
Plant cuticles are the interface between the above-ground
plant organs and the surrounding atmosphere. They form a
continuous layer of predominantly lipid material deposited
on the outer walls of epidermal cells (Fahn, 1990; Juniper,
1991). All plant cuticles contain waxes (the so-called
epicuticular waxes: EW), i.e. highly hydrophobic compounds that are deposited both within the cuticular matrix
and on its surface as an amorphous ®lm (Fahn, 1990;
Kolattukudy, 1996). This ®lm gives the cuticle its hydrophobic character that determines the extent of wettability of
the plant surface. Thus, the epicuticular wax layer prevents
the formation of stable, macroscopic water phases and,
hence, the germination of the spores of many plant pathogens (Allen et al., 1991; Juniper, 1991; Mendgen, 1996). It is
also a protective barrier against water loss by evaporation
and against loss of inorganic and organic constituents by
leaching from the interior of the plant tissues (Riederer and
MarkstaÈdter, 1996). Moreover, epicuticular waxes of higher
plants provide a mechanical obstacle that prevents the entry
of phytopathogenic bacteria and fungi (Mendgen, 1996). In
addition, this layer plays a signi®cant role in host-plant
recognition of certain fungi (Podila et al., 1993; Mendgen,
* For correspondence. Fax 003 1 5294286, e-mail [email protected]
0305-7364/01/050641+08 $35.00/00
1996) and insect herbivores (Eigenbrode, 1996; Kerstiens,
1996). The external layer of EW in¯uences both the reception
and penetration of incident radiation (Barnes and CardosoVilhena, 1996). The morphology, as well as the composition
of EW varies widely between species or cultivars (Hull et al.,
1975; Bianchi et al., 1993; Shepherd et al., 1995) and is also
a€ected by plant age and certain environmental factors,
such as heat, humidity and irradiance levels (Bianchi et al.,
1992; Riederer and MarkstaÈdter, 1996).
Karabourniotis et al. (2001) found that immersing leaves
of three representative plant species possessing morphologically di€erent stomatal complexes in chloroform for 30 s
(thus removing epicuticular waxes), signi®cantly reduced the
intensity of the blue ¯uorescence emitted from the guard
cells. It was highly likely, therefore, that the chloroform
rinses contained ¯uorescing compounds. The aim of our
study was to con®rm the presence of ¯uorescence compounds in chloroform leaf rinses of selected plant species
and to determine the chemical nature of these compounds.
M AT E R I A L S A N D M E T H O D S
Plant material
Leaves of Olea europaea and Prunus persica were collected
during summer 2000 from the experimental plantation of
# 2001 Annals of Botany Company
642
Liakopoulos et al.ÐPhenolics in Leaf Epicuticular Waxes
the Agricultural University of Athens, Greece. Leaves were
immediately wrapped in plastic bags and transferred to the
laboratory.
Preparation of epicuticular wax rinses
Leaves (total surface area 1011 cm2 for O. europaea and
1100 cm2 for P. persica) were rinsed with chloroform for
30 s at room temperature. Leaves of O. europaea were
dehaired using self-adhesive tape prior to the above
treatment to remove the trichome layer, which mainly
covers the abaxial surface. To prepare separate chloroform
rinses from the two leaf surfaces (adaxial and abaxial),
leaves of O. europaea and P. persica were rinsed with
chloroform using a teat pipette, ensuring that only one
surface was in contact with chloroform. Each leaf side of
O. europaea was rinsed with approx. 4 ml of chloroform
while 8 ml was used for each leaf side of P. persica. Rinsed
leaves were immediately scanned in a PC system and the
total leaf area was measured by standard image analysis
using Image Pro Plus for Windows, ver. 3.01 (Media
Cybernetics, Silver Spring, Maryland, USA). Chloroform
rinses were ®ltered through Whatman No. 3 ®lter paper and
concentrated in vacuum under a nitrogen stream at 30 8C.
Dry residues were weighed to determine wax yield, rediluted
in methanol (20 ml) and stored at 8 8C.
Measurement of emission spectra of the crude rinses
Emission spectra were taken from 376 to 600 nm
(excitation light 366 nm), using ®ltered chloroform rinses
or methanolic solutions of standard compounds in an FP920 ¯uorescence detector (Jasco Corporation, Tokyo,
Japan). Emission spectra of alkali treated samples were
taken by adding two drops of aqueous 10 % KOH solution
to the chloroform samples (2 ml) and mixing thoroughly.
Hydrolysis of epicuticular waxes
Aliquots (10 ml) containing the epicuticular waxes were
placed in a 100 ml ¯ask with 10 ml 8 N NaOH and kept for
1 h at 60 8C under a nitrogen stream. After hydrolysis,
samples were placed in an ice bath and 8 N HCl was slowly
added to neutralize the solution ( pH 7). Samples were
extracted three times with 15 ml ethyl acetate and the
combined extracts were concentrated in vacuum under a
nitrogen stream at 30 8C. Residues were rediluted in 2 or
3 ml 50 % methanol and stored at ÿ20 8C.
Chromatographic analysis of untreated and hydrolysed rinses
Analyses were performed in an HPLC system equipped
with an LG-980-02 tertiary low-pressure gradient unit, a
PU-980 pump, a UV-970 ultraviolet detector and an FP-920
¯uorescence detector connected downstream of the UV-970
(Jasco Corporation). Samples were ®ltered using RC
0.20 mm membrane ®lters (Lida Manufacturing Corp.,
Kenosha, Wisconsin, USA) and injected into an APEX
ODS 5 mm, 25.0 4.6 mm analytical column in line
with a 1 cm APEX ODS 5 mm guard column (Jones
Chromatography Limited, Mid Glamorgan, UK) using a
7725i injection valve (Rheodyne, Rohnhert Park,
California, USA) with a 20 ml sample loop. The analytical
column was kept at 30 8C using a column heater, Model
7971 (Jones Chromatography Limited). Chromatographic
solvents were of HPLC grade (LabScan Ltd., Dublin,
Ireland), formic acid was of analytical grade (Merck KGaA,
Darmstadt, Germany) and the mobile phase was degassed
with helium during analysis. Elution solvents were 5 %
aqueous HCOOH (A) and acetonitrile (B). The following
gradient elution was used: 95 : 5 (A : B), isocratic for 5 min;
gradient to 85 : 15 in 15 min; isocratic for 5 min; gradient to
77.5 : 22.5 in 10 min; gradient to 72.5 : 27.5 in 10 min;
gradient to 67.5 : 32.5 in 5 min; gradient to 55 : 45 in 5 min;
gradient to 50 : 50 in 5 min; gradient to 30 : 70 in
5 min; gradient to 0 : 100 in 5 min. Flow rate was
1.2 ml min ÿ1; detection wavelengths 300 nm (ultraviolet
detector); 430 nm ( ¯uorescence detector, using excitation
light at 365 nm). Chromatograms were recorded in a PC
system using Borwin Chromatographic Software, ver.
1.21.60 (JMBS Developments, Fontaine, France). Chromatograms from the ¯uorescence detector were corrected by
subtracting the corresponding blank taken with HPLC
grade 50 % MeOH, using the above software. Compounds
of interest were characterized by comparing their retention
times and UV-absorption spectra to those of standard
compounds (Extrasynthese S.A., Genay, France), as well as
by co-chromatography. Quantitative determination was
performed according to reference curves.
R E S U LT S A N D D I S C U S S I O N
Two species possessing hypostomatous leaves were chosen
to detect any di€erences in the phenolic constituents of the
rinses derived either from the adaxial (without stomata) or
the abaxial (with stomata) leaf surface. Guard cells of
O. europaea leaves emit a blue ¯uorescence following alkali
treatment, brighter than that of the surrounding epidermal
cells (see Fig. 1B in Karabourniotis et al., 2001). Fluorescence images from the abaxial surface of P. persica leaves
were more heterogeneous following alkali treatment
(Fig. 1B) since guard cells emitted blue ¯uorescence,
whereas epidermal cells emitted yellow-green ¯uorescence.
To con®rm that the ¯uorescing compounds were deposited
in the cuticular layer, P. persica leaves were immersed in
chloroform for 30 s to remove epicuticular waxes. This
treatment reduced the intensity of the blue ¯uorescence
emitted from the guard cells (Fig. 1C), as in the case of olive
leaves (see Fig. 1C in Karabourniotis et al., 2001).
Microscopic examination of the leaf surfaces of both
species showed no glands or glandular hairs, whose
secondary metabolites might be extracted by chloroform
and therefore alter the composition of the rinses.
Fluorescence emission spectra (Fig. 2A and B) of the
crude chloroform rinses of the leaves of both plants
con®rmed the occurrence of ¯uorescing compounds. In
O. europaea rinses, a maximum emission peak in the blue
(430±460 nm), as well as a minor peak in the green
(500±550 nm) region of the spectrum were observed,
whereas in P. persica there were two distinct peaks in the
Liakopoulos et al.ÐPhenolics in Leaf Epicuticular Waxes
Fluoresence intensity (cm–2 ml–1)
0.250
643
A
0.200
0.150
0.100
0.050
0.000
Fluoresence intensity (cm–2 ml–1)
0.250
B
0.200
0.150
0.100
0.050
0.000
Fluoresence intensity (µg–1 ml–1)
0.020
C
0.015
0.010
0.005
0.000
350
400
450
500
550
600
Wavelength (nm)
F I G . 2. A, Emission spectra of crude chloroform rinses from leaves of
Olea europaea upon excitation with 366 nm light. Adaxial surface:
untreated (ÐÐ), alkali treated (± ± ± ±); abaxial surface: untreated
(± ± ±), alkali treated (Ð Ð Ð Ð). Intensity values are normalized at
1 cm2 of leaf surface and 1 ml of rinse. B, Emission spectra of crude
chloroform rinses from leaves of Prunus persica upon excitation with
366 nm light, details as in A. C, Emission spectra in methanol of
p-coumaric acid (ÐÐ) and ferulic acid (± ± ± ±).
F I G . 1. Fluorescence micrographs of the abaxial surface of Prunus
persica leaves under UV radiation after di€erent treatments. A,
Untreated leaf; B, leaf immersed for 2 min in a solution of 10 %
KOH and washed with distilled water; C, leaf immersed for 2 min in a
solution of 10 % KOH, washed with distilled water and immersed in
chloroform for 30 s. In C note the reduction of the intensity of the blue
¯uorescence emitted from the guard cells, as well as of the blue-green
¯uorescence emitted from epidermal cells. Bar ˆ 100 mm.
blue and green regions. The rinses from the abaxial leaf
surface of O. europaea ¯uoresced more intensely than those
of the adaxial surface, while in P. persica the di€erence in
intensity between surfaces was negligible (Fig. 2A and B).
Alkali treatment of the chloroform samples caused an
increase in the emitted ¯uorescence, as well as a
644
Liakopoulos et al.ÐPhenolics in Leaf Epicuticular Waxes
C
UV detector response
A
Fluorescence detector response
0
10
20
30
40
50
60
0
B
0
10
20
10
20
30
40
50
60
30
40
50
60
D
10
20
30
40
50
60
Time (min)
0
Time (min)
F I G . 3. HPLC chromatograms of the phenolic constituents of the rinses of O. europaea leaves after alkaline hydrolysis (see Materials and
Methods). Chromatograms A and C were taken using a UV-detector at 300 nm and B and D using a ¯uorescence detector (excitation at 365 nm;
emission at 430 nm). A and B, Adaxial surface; C and D, abaxial surface. Arrow denotes ferulic acid elution peak; arrowhead denotes p-coumaric
acid elution peak. The response of each detector has been scaled so as to correspond to the same leaf area and the same sample volume for all
samples presented in the ®gure, as well as of those of Fig. 4. For chromatographic conditions see Materials and Methods.
bathochromic shift to higher wavelengths. This result
appeared in all rinses except that of the adaxial leaf surface
of P. persica (Fig. 2A and B). The appearance of the
emission spectra of untreated and alkali-treated samples was
consistent with the ¯uorescence images of the corresponding
leaf surfaces (with or without alkali treatment) of both
plants (Fig. 1, see also Fig. 1 in Karabourniotis et al., 2001).
Recently, it was found that ¯uorescence yield index values
(i.e. the emission peak energy divided by the total excitation
energy) of abaxial leaf surfaces obtained from 35 species
tended to be higher than the corresponding values of adaxial
surfaces (Johnson et al., 2000).
Surprisingly, HPLC chromatographic analysis of the
chloroform rinses, from either the adaxial or abaxial leaf
surface of both plants did not show any detectable peak
using UV absorbance or ¯uorescence detection (data not
shown). However, a number of peaks appeared in the
chromatogram after alkaline hydrolysis (see Materials and
Methods) of the samples. Ferulic acid and p-coumaric acid,
as well as a number of unidenti®ed compounds, were
constituents of the rinses of both plants examined (Figs 3A,
C and 4A, C). This result indicates that these compounds
are tightly bound to the epicuticular waxes of the leaves of
these plants. Ferulic and p-coumaric acids, and a number of
unidenti®ed peaks, were also detected by the ¯uorescence
detector (Figs 3B, D and 4B, D) in spite of the low pH value
of the mobile phase (see Materials and Methods). In
addition, the emission spectra, especially of the rinse from
the abaxial surface of olive leaves (Fig. 2A) resembled that
obtained using ferulic acid as a reference (Fig. 2C).
Hydroxycinnamic acids, such as ferulic and p-coumaric
acid, are important structural components which serve to
cross-link polymers in plant cell walls (Kroon and Williamson, 1999). Ferulic acid is considered to be the main
¯uorescent compound responsible for the blue ¯uorescence
emitted by cell walls of some species (Harris and Hartley,
1976; Lichtenthaler and Schweiger, 1998) and for the bluegreen ¯uorescence emitted by the epidermis of sugar beet
leaves (Morales et al., 1996). Methanolic solutions of ferulic
acid exhibit a distinct blue ¯uorescence emission between
440 and 455 nm upon UV-A excitation (Lichtenthaler and
Schweiger, 1998, see also Fig. 2C). Under the same
conditions of excitation, p-coumaric acid showed only a
weak blue ¯uorescence (Fig. 2C). However, alkali treatment
induced a bathochromic shift of the ¯uorescence emission
spectra of both compounds (Ibrahim and Barron, 1989).
Similar behaviour was also exhibited by the crude rinses
(Fig. 2A and B). Riley and Kolattukudy (1975) found that
p-coumaric acid and smaller amounts of ferulic acid were
covalently attached to fruit cuticles, as well as to those of
tomato and apple tree leaves. The amount of covalently
attached coumaric acid, as well as of some ¯avanoids, in the
cutin of the tomato fruit increased during fruit development
(Hunt and Baker, 1980).
Comparison of HPLC pro®les between adaxial and
abaxial chloroform rinses of olive leaves did not reveal
Liakopoulos et al.ÐPhenolics in Leaf Epicuticular Waxes
A
645
UV detector response
C
Fluorescence detector response
0
10
20
30
40
50
60
0
B
0
10
20
30
40
50
60
10
20
30
40
50
60
D
10
20
30
40
50
60
Time (min)
0
Time (min)
F I G . 4. HPLC chromatograms of the phenolic constituents of the rinses of P. persica leaves. Details as for Fig. 3.
any signi®cant qualitative di€erences (compare Fig. 3A
with C). In contrast, dramatic qualitative changes between
the corresponding samples of P. persica leaves were
observed (compare Fig. 4A with C). The complex HPLC
pro®le of the abaxial surface of P. persica leaves and the
absence of corresponding phenolic compounds in the
adaxial surface may re¯ect the heterogenous ¯uorescence
image produced by the abaxial surface under the ¯uorescence microscope and may also re¯ect the fact that the
adaxial surface of P. persica lacked ¯uorescence emission
even when treated with alkali (data not shown). It is
possible, therefore, that wax-bound, yellow-green ¯uorescing compounds ( probably ¯avonoids and other related
substances) located in the epicuticular layer over epidermal
and/or guard cells, were extracted by chloroform. The
occurrence of ¯avonoids or related substances was also
supported by HPLC analysis via the elution and spectral
characteristics of numerous, still unidenti®ed, compounds
in the rinse from the abaxial surface (data not shown).
The two species examined showed distinct di€erences in
the characteristics of the epicuticular layer covering either
the adaxial or abaxial leaf surface. Chloroform rinses
derived from the xeromorphic leaves of O. europaea contained signi®cantly greater amounts of wax, as well as of
p-coumaric and especially ferulic acid, than rinses from
P. persica leaves. Moreover, the amount of wax on the
adaxial surface of olive leaves was greater than that on the
abaxial surface (Fig. 5A). The amount of wax from
the adaxial surface was similar to that reported by Leon
and Bukovac (1978). However, Bianchi et al. (1993) reported
two-fold more wax after immersing whole, intact olive leaves
in chloroform. This contradiction may be due to di€erences
between olive varieties and/or extraction procedures. Unlike
olive, the amount of wax from the abaxial surface of
P. persica leaves was at least four-fold higher than that from
the adaxial surface. According to Riederer and Schneider
(1989), the qualitative, as well as the quantitative composition of epicuticular waxes may vary considerably between
abaxial and adaxial leaf surfaces.
The concentration of p-coumaric acid and ferulic acid
(expressed as mg per mg of waxes) was higher in rinses from
the adaxial surface than from the abaxial surface of
P. persica leaves. In O. europaea, the concentration of pcoumaric acid was higher in leaf rinses from the adaxial
compared to the abaxial surface, while the concentration of
ferulic acid was much higher in rinses from the abaxial leaf
surface (Fig. 5B). These results show that the concentration
of phenolic compounds in the epicuticular layer is not
determined solely by corresponding di€erences in wax
deposition but also by speci®c variations of the proportion
of phenolic compounds involved in the construction of the
epicuticular layer of the di€erent leaf surfaces. The
concentration of ferulic acid (expressed as ng per leaf
surface area) was higher in rinses from the abaxial surface
(with stomata) than that from the adaxial one, in both
plants. Furthermore, the highest concentration of ferulic
acid ( per leaf surface area) was found in the abaxial surface
of O. europaea (Fig. 5C), which exhibited the highest
¯uorescence emission under the microscope, with or without
alkali treatment (compare Fig. 1A or B in Karabourniotis
et al., 2001 with Fig. 1A or B). Moreover, the only di€erence
between adaxial and abaxial surface images of O. europaea
646
Liakopoulos et al.ÐPhenolics in Leaf Epicuticular Waxes
Amount per surface area (µg cm–2)
300
A
250
200
150
100
50
0
adaxial
abaxial
adaxial
abaxial
adaxial
abaxial
adaxial
abaxial
adaxial
abaxial
adaxial
abaxial
Olea europaea
B
Concentration (µg mg
–1
)
1.0
Prunus persica
0.8
0.6
0.4
0.2
0.0
Olea europaea
Prunus persica
Amount per surface area (ng cm–2)
175
C
150
50
40
30
20
10
0
Olea europaea
Prunus persica
Species / leaf surface
F I G . 5. A, The amount of epicuticular waxes extracted either from
adaxial or abaxial surfaces of the leaves of Olea europaea and Prunus
persica. B, Concentration of p-coumaric acid (j) and ferulic acid (h)
expressed per unit of epicuticular wax mass, for the same samples. C,
Concentration of p-coumaric and ferulic acid expressed per unit leaf
area of the corresponding leaf surface for the same samples.
(without alkali treatment) was the intense ¯uorescence
emitted by guard cells. The corresponding di€erence in the
compounds emitting intense ¯uorescence revealed by HPLC
analysis was the presence of much greater amounts of ferulic
acid in the abaxial surface. It is possible, therefore, that
ferulic acid is the main compound responsible for the blue
¯uorescence of guard cells of O. europaea, taking into
account its particular ¯uorescence characteristics and the
reduction in the intensity of the blue ¯uorescence emitted by
guard cells upon chloroform treatment.
The concentration of p-coumaric acid (expressed as ng
per leaf surface area), was higher in rinses from the abaxial
surface than from the adaxial surface of P. persica leaves,
whereas the opposite trend was observed in O. europaea
leaves (Fig. 5C). Thus, rinses from the adaxial surface of
olive leaves, possessing lower concentrations of ferulic acid,
but higher concentrations of p-coumaric acid compared to
rinses from the abaxial surface, showed a lower ¯uorescence
intensity than the abaxial surface (Fig. 2A). Moreover, the
¯uorescence spectra of rinses from abaxial surfaces of olive
leaves resembled those of reference p-coumaric acid
(compare Fig. 2A with C).
The occurrence of phenolic compounds in the epicuticular layer covering guard cells may be related to defence
against biotic as well as abiotic factors. Since stomata
constitute an entry to the leaf interior for pathogens that are
able to grow through the pores, it is possible that defensive
metabolites may be deposited on the surface waxes covering
guard cells. Inoculation of leaves of Vitis rotundifolia with
the downy mildew fungus, Plasmopara viticola, caused the
formation of phenolic compounds in guard cells (Dai et al.,
1995). Secondary defensive substances may be present in
the wax layer of the leaves of a number of plants (Juniper,
1991). The phenolic constituents of the epicuticular layer,
probably esteri®ed to the waxes, might be released by the
hydrolytic action of phytopathogenic fungi, providing
additional protection under these conditions (Kolattukudy,
1981). The penetration of infection hyphae is also inhibited
by phenolic acids found in waxy coverings of apple tree
leaves (Martin et al., 1957). Low molecular weight phenolic
compounds, such as ferulic acid and p-coumaric acid, are
active in the defensive response of plants against pathogens
(Nicholson and Hammerschmidt, 1992; Bennett and
Wallsgrove, 1994). Ferulic acid is also an e€ective feeding
deterrent (Bennet and Wallsgrove, 1994). The chemical
nature of the surface is also important in the cuticle-fungal
interaction and may play a signi®cant role in site
recognition processes (Allen et al., 1991).
Both the epicuticular waxes and the cuticular membrane
may a€ect the penetration of UV radiation into the
mesophyll. A number of recent studies showed that cuticles
isolated from a range of leaves and fruits exhibited strong
absorbance in the UV region of the spectrum (Scherbatskoy, 1994; Krauss et al., 1997). UV absorbance by leaf wax
extracts seems to be species-speci®c (Bornman and Vogelman, 1988; Robinson et al., 1993). The results of the present
study showed that the concentration of the UV absorbing,
wax-bound phenolic constituents was not only speciesspeci®c, but may also di€er between adaxial and abaxial
leaf surfaces of the same plant. The occurrence of phenolic
constituents in the epicuticular wax layer, especially in that
covering guard cells, may also be related to a UV protective
function of these compounds. The mechanism of stomatal
movements of a number of plant species appears to be
Liakopoulos et al.ÐPhenolics in Leaf Epicuticular Waxes
sensitive to large ¯uxes of UV-B radiation (NogueÂs et al.,
1999). However, there are indications that guard cells
possess special UV protective potential. In Smilacina
stellata, an herbaceous species, UV radiation transmittance
of leaf epidermes is relatively high through stomatal pores,
but relatively low through stomatal guard cells (Day et al.,
1993). On the other hand, the percentage increase of
hydroxycinnamic derivatives under elevated UV-B radiation is observed in the abaxial epidermis of Brassica napus
leaves, in comparison to that in the adaxial epidermis and
mesophyll (Bornman, 1999).
Recent studies have indicated that UV-B radiation may
have marked e€ects on the production and/or chemical
composition of the epicuticular waxes of some, but not all,
wild and crop plants (Gordon et al., 1998; Manetas, 1999;
Pilon et al., 1999). Therefore, it will be of interest to
investigate the e€ect of increased UV-B radiation on the
phenolic constituents of the epicuticular wax layer.
AC K N OW L E D GE M E N T S
The authors thank Professor M. Polyssiou for helpful
suggestions on the analytical procedure.
L I T E R AT U R E C I T E D
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