Very long chain alkylresorcinols accumulate in the intracuticular wax of rye (Secale cereale L.) leaves near the tissue surface

Article abstract of DOI:10.1016/j.phytochem.2007.12.008

Alkylresorcinols (ARs) are bioactive compounds occurring in many members of the Poaceae, likely at or near the surface of various organs. Here, we investigated AR localization within the cuticular wax layers of rye (Secale cereale) leaves. The total wax m

Full text of DOI:10.1016/j.phytochem.2007.12.008

Available online at www.sciencedirect.com
PHYTOCHEMISTRY
Phytochemistry 69 (2008) 1197–1207
www.elsevier.com/locate/phytochem
Very long chain alkylresorcinols accumulate in the intracuticular
wax of rye (Secale cereale L.) leaves near the tissue surface
Xiufeng Ji, Reinhard Jetter *
Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, Canada V6T 1Z4
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, Canada V6T 1Z1
Received 10 September 2007; received in revised form 7 December 2007
Available online 29 January 2008
Abstract
Alkylresorcinols (ARs) are bioactive compounds occurring in many members of the Poaceae, likely at or near the surface of various
organs. Here, we investigated AR localization within the cuticular wax layers of rye (Secale cereale) leaves. The total wax mixture from
both sides of the leaves was found to contain primary alcohols (71%), alkyl esters (11%), aldehydes (5%), and small amounts (<3%) of
alkanes, steroids, secondary alcohols, fatty acids and unknowns. A homologous series of ARs (3%) was identified by GC–MS and com-
parison with a synthetic standard of nonadecylresorcinol. The alkyl side chains of the wax ARs contained odd numbers of carbons rang-
ing from C19 to C27, with a prevalence of C21, C23 and C25. Waxes from both sides of the leaf, analyzed separately in a second experiment,
comprised the same compound classes in similar relative amounts and with similar homolog patterns. Finally, the epicuticular and
intracuticular wax layers were sampled separately from the abaxial side of the leaf. While ARs accounted for 2% of the intracuticular
wax, they were not detectable in the epicuticular wax. The intracuticular wax was also slightly enriched in steroids, whereas the epicu-
ticular layer contained more primary alcohols. All other wax constituents were distributed evenly between both wax layers.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Rye; Secale cereale; Gramineae; Epicuticular wax; Intracuticular wax; Chain lengths; GC–MS
1. Introduction
in eleven families (Ginkgoaceae, Anacardiaceae, Protea-
ceae, Myrsinaceae, Primulaceae, Myristicaceae, Fabaceae,
Asteraceae, Iridaceae and Poaceae) (Kozubek and Tyman,
1999). They were initially isolated from fruits, seeds and
various senescent organs, and only later were they also
detected in green leaves and stems.
There is indirect evidence that ARs accumulate at or
near the surface of diverse organs, where they exhibit anti-
bacterial and antifungal activities. ARs are present in the
outer (aleurone and pericarp) regions of wheat and rye
grains, resulting in relatively high AR concentrations in
bran and shorts milling fractions as compared to flours
(Ross et al., 2003). Extracts of thin fruit peels of Mangifera
indica (mango), were reported to contain ARs (conferring
resistance to Alternaria alternata) in concentrations twelve
times higher than those of the flesh (Droby and Prusky,
1987). More interestingly, the outer layer of mango flesh
accumulated high AR concentrations after peeling, while
1,3-Dihydroxy-5-alkylbenzenes, also known as 5-
alkylresorcinols (ARs), are potent bioactive secondary
metabolites that have been shown, e.g., to affect bilayer
membranes due to their amphiphilic properties, and to
interfere with growth regulation of cells through interac-
tion with nucleic acids and enzymes (Kozubek and Tyman,
1999; Prakash Chaturvedula et al., 2002; Kozubek and
Demel, 1980). ARs typically occur as homologous series
with alkyl chain lengths ranging from C5 to C29 (Kozubek
and Tyman, 1999). The alkyl side chains have predomi-
nantly odd numbers of carbons, which is significant with
regard to AR biogenesis. ARs occur in bacteria, animals,
fungi and plants, where they have to date been described
*
Corresponding author. Tel.: +1 604 822 2477; fax: +1 604 822 6089.
E-mail address: jetter@interchange.ubc.ca (R. Jetter).
0031-9422/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.12.008

1198
X. Ji, R. Jetter / Phytochemistry 69 (2008) 1197–1207
the flesh underneath did not. ARs are also found in the
seed covers of Myristica fragrans (nutmeg), where they
have been reported to confer antibacterial activity against
Staphylococcus aureus (Orabi et al., 1991). Finally, the fruit
shells of Anacardium occidentale (cashew nut) are known to
accumulate ARs that cause severe dermatitis when the fruit
surfaces are brought into contact with human skin (Nogue-
ira Diogenes et al., 1996). Since most occurrences of ARs
are in tissues that are exposed at the plant surface, it may
be speculated that the respective ARs are produced by epi-
dermal cells and are exposed at the surface to protect the
interior tissue. However, direct evidence for the surface
accumulation of ARs is scarce.
The surface of primary above-ground plant organs is
covered with a cuticle, a protective lipid structure sealing
the tissue against the environment. The cuticle serves as
the major barrier preventing nonstomatal water loss, helps
to protect plant surfaces against pathogens and ultraviolet
radiation, and affects plant-insect interactions (Jenks et al.,
1994; Holmes and Keiller, 2002; Eigenbrode and Jetter,
various barley (Hordeum vulgare) cultivars (Garcia et al.,
1997). Unfortunately, wax analyses have not been carried
out for diverse other species known to synthesize ARs.
Where wax analyses have been carried out for other AR-
containing species, these compounds were not reported
for the wax mixtures. For example, rye (Secale cereale
L.) contains relatively high amounts of ARs (Montsant
et al., 2005; Magnucka et al., 2007), but ARs are not men-
tioned in reports on the cuticular waxes from this species
(Streibl et al., 1974; Tulloch and Hoffman, 1974). How-
ever, this is not sufficient evidence against the accumula-
tion of cuticular ARs in this species, as the compounds
might have been overlooked in the early wax analyses or
AR accumulation might be restricted to certain parts of
the plants that were not investigated in the wax analyses.
The AR reports are referring to rye grains and seedlings,
whereas the wax analyses were performed on leaves and
straw (i.e. leaves, stems and spikes). It is therefore not
clear whether ARs in these instances accumulated partially
or even exclusively in this lipophilic compartment, and
whether they are targeted to the cuticle to serve biological
functions at the tissue surface. It is also not clear how ARs
might be partitioned between the epi- and intracuticular
wax layers.
The goal of the present investigation was to localize ARs
in cuticular wax mixtures of Poaceae. Preliminary experi-
ments had shown that all leaves of rye contain relatively
high amounts of ARs, as compared with other species
and organs. Here, we performed detailed chemical analyses
to address the questions: (1) which VLC compounds are
present in the waxes of rye leaves, (2) whether the abaxial
and adaxial waxes differ, (3) whether the epicuticular and
intracuticular wax layers have different composition, (4)
whether ARs exist in the leaf cuticular wax, and (5)
whether ARs are restricted to one side of the leaf and/or
to either the epi- or intracuticular wax layers.
2002; Vogg et al., 2004; Furstner et al., 2005). Plant cuticles
¨
consist of cutin, an insoluble polyester lattice typically con-
stituting 40–80% of the cuticle mass, and of soluble cuticu-
lar waxes. The latter are complex mixtures of very long
chain (VLC) fatty acid derivatives, typically unbranched
and fully saturated alcohols, aldehydes, ketones, fatty
acids, esters and alkanes (Jetter et al., 2006). Each of these
compound classes comprises a homologous series with
chain lengths ranging from C20 to C36 (esters from C36 to
C70). Depending on the plant species and organ, wax mix-
tures may also contain cyclic compounds such as triterpe-
noids and phenylpropanoids (Wen et al., 2006).
Part of the cuticular wax is embedded within the poly-
mer network of cutin, and is designated as intracuticular
wax. An additional layer of so-called epicuticular wax is
deposited onto the outer surface of the cutin matrix, where
it forms the true surface of the tissue (Jeffree, 1986). Both
epi- and intracuticular waxes can be extracted together in
one step by dipping the intact surface of an organ into
organic solvents. Alternatively, both wax layers can be
sampled separately by first employing adhesives to strip
the epicuticular material, and then organic solvents to
extract the remaining intracuticular wax (Jetter et al.,
2000). For a number of species, the two wax layers were
found to have drastically differing composition, with some
constituents of the mixture being enriched in the epi- and
others in the intracuticular wax (Jetter et al., 2006). For
example, selective analyses of both wax layers on Taxus
baccata needles showed that relatively polar components
tended to accumulate in the intracuticular wax, and that
aromatics (phenylpropanoids and phenylbutanoids) were
entirely restricted to this layer (Wen et al., 2006).
2. Results and discussion
The goal of the present investigation was to analyze the
chemical composition of the cuticular waxes on the adaxial
and abaxial sides of Secale cereale leaves, with special
emphasis on the localization of alkylresorcinols in the epi-
cuticular and intracuticular wax layers. To minimize bio-
logical variation, analyses were restricted to well-defined
segments of the second leaf. Preliminary results had shown
that differentiation and growth of epidermal cells had
ceased in the leaf zone 10 cm above the point of emergence,
resulting in relatively constant wax mixtures over time.
Three sets of experiments were carried out to describe the
wax composition with three different levels of spatial reso-
lution. In the first experiment, total wax mixtures were
extracted from both sides of the leaves together, in order
to acquire overall compositional data comparable with
those reported for other grass species. The second and third
experiments were aimed at distinguishing the composition
The occurrence of ARs near the surface of certain plant
species and organs, together with the lipophilic character
of the AR side chains, raises the question whether the
ARs are localized in the cuticular waxes in these systems.
Cuticular ARs have been reported only once for seeds of

X. Ji, R. Jetter / Phytochemistry 69 (2008) 1197–1207
1199
of waxes from both sides of the leaves, and from the epi-
and intracuticular wax layers, respectively.
rye leaves (Tulloch and Hoffman, 1971; Bianchi et al.,
1979; Reynhardt and Riederer, 1994; Koch et al., 2005).
The cuticular waxes of rye have not been studied in similar
detail to date. Wax extracted from rye straw contained
alkanes, primary alcohols, fatty acids and esters together
with high amounts of b-diketones not found in our study
(Streibl et al., 1974). Similar results were reported for
wax mixtures extracted from all leaves together (Tulloch
and Hoffman, 1974). Overall, the discrepancies between
our results and the previous reports cannot be attributed
to variability between rye cultivars alone, but are likely
to also reflect drastic differences between the wax composi-
tions of various organs of rye. b-diketones are the predom-
inant wax constituents of barley spikes and stems (von
Wettstein-Knowles, 1982; Richardson et al., 2007), as well
as wheat flag leaves (Tulloch, 1973). By analogy, it seems
plausible that the b-diketones previously reported for rye
waxes originated from these organs rather than the lower
leaves investigated here.
In addition to the typical wax constituents described
above, the leaf surface extracts of S. cereale were found
to contain a number of compounds that had not previously
been reported from leaf cuticular waxes. Based on their GC
retention behavior and MS characteristics, they were
recognized as a homologous series of five compounds
differing by –CH2ACH2– units. The corresponding
TMSi derivatives showed MS fragments of m/z 73, 268,
281 characteristic for alkylresorcinols (ARs) (Linko et al.,
2002; Ross et al., 2003), together with molecular ions
In the initial experiment, the total leaf wax was extracted
from the entire exposed leaf surface including both the
adaxial and the abaxial sides. The total wax coverage on
the leaf was 12.2 1.5 lg/cm2 (Fig. 1). The mixture con-
tained large amounts of primary alcohols (71%) together
with alkyl esters (11%), aldehydes (5%), alkanes (3%), ste-
roids (0.3%), and traces of secondary alcohols as well as
fatty acids. The fatty acid, primary alcohol and aldehyde
fractions contained homologous series of predominantly
even carbon-numbered compounds, with chain lengths
ranging between C20–C34, C22–C30 and C26–C32, respec-
tively (Table 1). The C26 homologs strongly predominated
in all three series (Fig. 2), making hexacosanol the single
most abundant constituent with 69% (8.7 1.3 lg/cm2)
in the rye leaf total wax mixture. The alkane fraction was
found to contain a homologous series ranging from C27
to C33 (Table 1), with a relatively broad chain length distri-
bution of predominantly odd-numbered compounds and a
maximum at C31 (Fig. 2). The alkyl esters were identified as
a homologous series ranging from C40 to C52 (even num-
bers only) with a maximum at C44 (Table 2).
Our findings for rye wax are similar to the cuticular wax
compositions reported for other Poaceae species. Rice
(Oryza sativa), wheat (Triticum aestivum and T. durum),
barley (Hordeum vulgare) and maize (Zea mays) waxes
had consistently been found to contain alkanes, primary
alcohols, fatty acids and aldehydes similar to those on
10
8
Total
Adaxial
Abaxial
6
4
2
0
Fig. 1. Compound class composition of cuticular wax mixtures from the second leaf of rye. The coverages of all compound classes are given as mean
values (n = 6) and SD for the total wax extracted from both sides of the leaf together, and for the wax extracted from the adaxial and abaxial sides
separately.

1200
X. Ji, R. Jetter / Phytochemistry 69 (2008) 1197–1207
Table 1
Very long chain compounds identified in rye leaf waxes
Compound class
Fatty acids
Chain length
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
C31
C32
C33
C34
To
Ad
Ab
Ep In
To
Ad
Ab
Ep In
To
Ad
Ab
Ep In
To
Ad
To
Ad
Ab
Ep In
To
Ad
Ab
To
Ad
Ab
To
Ad
Ab
Ab
Ep In
Primary alcohols
Aldehydes
To
Ad
Ab
Ep In
To
Ad
Ab
To
Ad
Ab
To
Ad
Ab
To
Ad
Ab
To
Ad
Ab
To
Ad
Ab
Ep In
To
Ad
Ab
Ep In Ep In Ep In Ep
Ep In Ep
To
Ad
To
Ad
Ab
Ep In
To
Ad
Ab
To
Ad
Ab
Ab
Ep In
Alkanes
To
Ad
Ab
To
Ad
Ab
To
Ad
Ab
To
Ad
Ab
To
Ad
To
Ad
Ab
Ep In Ep In
Ep In Ep In Ep In Ep In Ep In
Secondary alcohols
To
Ad Ab
Ep In
(OH at C14–C17
)
Alkylresorcinols
To
Ad
Ab
In
To
Ad
Ab
In
To
Ad
Ab
In
To
Ad
Ab
In
To
Ad
Ab
In
Wax mixtures were sampled by extracting both sides of the leaf together (To), by extracting the adaxial (Ad) or the abaxial (Ab) sides selectively, or by
removing the epicuticular (Ep) and the intracuticular (In) wax layers consecutively from the abaxial side. Predominant homologs are highlighted in bold
face.
[C6H3(OTMSi)2(CH2)nCH3]+ and corresponding frag-
ments [M–15]+ indicating the loss of a methyl group. To
verify whether these wax constituents were indeed ARs,
one representative of the homologous series was synthe-
sized. This standard of 1,3-dihydroxy-5-nonadecylbenzene
(1; AR 19:0) (Fig. 3) was found to have identical mass spec-
tral characteristics as the wax compounds. Furthermore,
the standard co-eluted with one of the rye wax constituents
under the GC conditions used. This finding not only con-
firms the exact alkyl chain length, but also the isomer con-
figuration of the wax ARs. It had previously been shown
that various isomers of comparable phenolics, including
variation in both side-chain and ring-positional configura-
tion, have sufficiently different physical properties to be
separated by GC (Fritz and Moore, 1987). Thus, even
though individual AR isomers may not be distinguished
by MS alone, they can usually be identified by GC–MS
co-injection experiments. GC–MS comparison with the
authentic standard therefore unambiguously established
the structure of one rye wax constituent as AR 19:0 (1).
Based on the equal distances between GC peaks, all the
other rye wax constituents with identical MS characteristics
were identified as homologous ARs with alkyl chain
lengths C19 to C27 (1–5) (Table 1, Fig. 3). The series of
ARs was dominated by homologs with odd-numbered
alkyl side chains, mainly by ARs 21:0 (2), 23:0 (3) and
25:0 (4) (Fig. 4). Overall, the AR fraction contributed
0.3 0.1 lg/cm2 to the wax coverage on the leaves,
accounting for approximately 3% of the total wax mixture.
After identification of the ARs, only 2% of the wax mixture
remained unidentified.
To test whether ARs are restricted to the cuticular wax
of rye leaves or whether similar AR homologs also occur in
the interior of the organ, surface waxes were first removed
and then standard protocols for analysis of internal ARs
were applied. ARs were quantified against an internal
standard of synthetic AR13:0 (6) using the intensity of
the characteristic MS fragment (m/z 268). Internal AR
amounts were found to be 9 lg/g fresh weight, as com-
pared to 135 lg/g of cuticular ARs. Cuticular ARs thus
accounted for approximately 94% of the total AR contents
of the leaf.
In previously published analyses of rye waxes, ARs were
not reported (Streibl et al., 1974; Tulloch and Hoffman,
1974). However, ARs had been detected in total lipid
extracts from various organs of S. cereale. Rye grains were
found to contain 559 lg/g dry weight of AR with alkyl
chain lengths ranging from C17 to C25 (odd numbers only)
and a strong predominance of ARs 17:0, 19:0 and 21:0
(Montsant et al., 2005). In rye seedlings, Magnucka et al.
(2007) detected 3.1 lg/g dry weight of ARs 15:0 to 25:0,
with AR 17:0, AR 19:0 and AR 23:0 as the main homologs.
Unfortunately, it is not clear whether these ARs were
located in the cuticular waxes of respective organs, or

X. Ji, R. Jetter / Phytochemistry 69 (2008) 1197–1207
1201
60
50
40
30
20
10
0
100
A
Total
B
Adaxial
80
Abaxial
60
40
20
0
20 22 24 26 28 30 32 34
Chain length
22
24
26
28
30
Chain length
60
50
40
30
20
10
0
100
80
60
40
20
0
D
C
26
28
30
32
27
28
29
30
31
32
33
Chain length
Chain length
Fig. 2. Chain length distributions of individual wax components in the total, adaxial and abaxial wax mixtures on the second leaf of rye. Percentages of
individual homologs within the series of (A) fatty acids, (B) primary alcohols, (C) aldehydes and (D) alkanes are shown as means (n = 6) with SD.
Table 2
Homolog and isomer composition of very long chain alkyl esters identified in rye leaf waxes
Ester chain length
C40
Chain lengths of esterified acids
C14
C16
C18
C20
C22
C24
C26
To
To
To
Ad Ab
Ep In
To
Ad Ab
Ep In
Ad Ab
Ep In
Ad Ab
Ep In
C42
C44
C46
C48
To
Ad Ab
Ep In
To
Ad Ab
Ep In
To
Ad Ab
Ep In
To
Ad Ab
Ep In
To
Ad Ab
Ep In
To
Ad Ab
Ep In
To
Ad Ab
Ep In
To
Ad Ab
Ep In
To
Ad Ab
Ep In
Wax mixtures were sampled by extracting both sides of the leaf together (To), by extracting the adaxial (Ad) or the abaxial (Ab) sides selectively, or by
removing the epicuticular (Ep) and the intracuticular (In) wax layers consecutively from the abaxial side. Predominant acyl homologs are highlighted in
bold face.
whether they had accumulated partially or exclusively in
the interior tissues. Future AR analyses of these organs
should distinguish between these possibilities. Interestingly,
the AR mixtures from rye grains and seedlings both had
chain length ranges starting and ending with lower
homologs than the leaf cuticular ARs. The homologous
series of leaf ARs was also found to peak at longer chain
lengths than those of the other organs.
The identification of ARs in the total wax from rye
leaves raised the question whether these compounds are
present in both the adaxial and abaxial wax mixtures. A
second experiment was consequently designed to separately

1202
X. Ji, R. Jetter / Phytochemistry 69 (2008) 1197–1207
OH
R
HO
R*
Name
Number in text
Abbreviation
C19H39
C21H43
C23H47
C25H51
C27H55
C13H27
5-Nonadecylresorcinol
5-Heneicosylresorcinol
5-Tricosylresorcinol
5-Pentacosylresorcinol
5-Heptacosylresorcinol
5-Tridecylresorcinol
1
2
3
4
5
6
AR19:0
AR21:0
AR23:0
AR25:0
AR27:0
AR13:0
* R are n-alkyl chains.
Fig. 3. Structures of alkylresorcinols identified in rye leaf wax (1–5) and synthesized as standards for structure elucidation (1) as well as quantification (6).
50
Total
Adaxial
Abaxial
40
30
20
10
0
19
21
23
25
27
Alkyl chain length
Fig. 4. Chain length distributions of alkylresorcinols in the total, adaxial and abaxial wax mixtures on the second leaf of rye. Percentages of individual
homologs within the fraction are shown as means (n = 6) and SD.
analyze the waxes from both sides of the S. cereale leaves.
To this end, a method was used that had previously been
established for the separate extraction of waxes from both
sides of gymnosperm needles (Wen et al., 2006). It relies on
gentle brushing of one leaf surface with CHCl3–soaked fab-
ric glass and collecting the organic wax solutions for GC–
MS analyses. Preliminary experiments, in which the rye leaf
surfaces were brushed 20 times, yielded only approximately
two thirds of the total wax amounts present. After brush-
ing for more than 60 times the extracts started to appear
green, indicating contamination by chlorophyll and possi-
bly also other lipids from internal tissues. Therefore, a
standard protocol limited to 60 times brushing was used
to extract the surface waxes from both sides of rye leaves.
It yielded 12.1 2.5 lg/cm2 and 11.5 2.0 lg/cm2 from
the adaxial and abaxial surfaces, respectively. These find-
ings correspond to an overall average wax coverage of
11.8 3.2 lg/cm2, which is not significantly different from
the total wax load found in our first experiment. It can be
concluded that brushing of both sides of rye leaves with
fabric glass allowed the separate and exhaustive sampling
of adaxial and abaxial cuticular waxes.
All the compound classes previously identified in the
total wax mixture from entire rye leaves (see above) were
also detected in the separate extracts from both the adaxial
and abaxial sides (Fig. 1). There were no significant differ-
ences between the compound class percentages on both
sides of the leaves, and between the homolog patterns

X. Ji, R. Jetter / Phytochemistry 69 (2008) 1197–1207
1203
within these classes (Tables 1 and 2, Fig. 2). ARs were
found to have approximately equal coverages of
0.2 0.1 lg/cm2 on the abaxial side of the leaf (accounting
for 1.4 0.4% of the wax mixture) and of 0.3 0.1 lg/cm2
on the abaxial side (accounting for 2.2 0.5% of the wax).
Similarly, the chain length distributions in the AR fractions
from both sides of the leaves were found to match closely
(Table 1, Fig. 4).
Overall, our results show that rye leaves are covered by
adaxial and abaxial cuticles with relatively similar wax
composition. They are thus an example where the two sur-
faces of the same organ, even though they have physio-
logically and ecologically different significance, share the
same overall makeup. Separate analyses of waxes from
adaxial and abaxial leaf sides had previously been per-
formed for relatively few other species, and in many cases
marked differences in composition had been reported.
For example, the adaxial wax of Pisum sativum was found
to be dominated by primary alcohols, whereas the corre-
sponding abaxial wax contained large amounts of alkanes
(Holloway et al., 1977; Eigenbrode et al., 1998; Gniwotta
et al., 2005).
A third experiment was carried out to investigate
whether compositional differences existed between the
intracuticular and the epicuticular wax layers on rye leaves.
In particular, this was to address the question whether ARs
are localized in the inner wax layer or exposed at the very
surface, or whether they occur throughout the cuticular
wax. Since no pronounced differences had been found
between the compositions of the adaxial and abaxial waxes,
this last experiment focused only on the latter side of the
rye leaf.
Gum arabic was used as an adhesive for the mechanical
sampling of the surface wax layer, and could be applied to
the same leaf four times without damaging the abaxial
surface. The consecutive glue treatments yielded slightly
decreasing wax amounts of 2.3 1.0, 2.3 0.5, 2.2 0.5,
and 1.7 0.4 lg/cm2. Statistical analyses showed no signif-
icant difference between the cumulative wax yields after the
3rd and 4th treatments (ANOVA, N = 6, P = 0.066), indi-
cating that the four successive treatments together had
exhaustively removed the mechanically accessible wax and
that a fifth gum arabic treatment would not have yielded
more material. Similar to previous studies, it can be con-
cluded that the wax sampled in this way had been located
outside the mechanically resistant cutin matrix, and hence
comprised the complete epicuticular wax layer. When the
leaf surface was, after four treatments with gum arabic,
finally extracted with CHCl3, substantial quantities of wax
could be retrieved. The corresponding wax yield after the
final chloroform extraction (6.0 2.0 lg/cm2) differed
significantly from that of the 4th adhesive treatment
(ANOVA, N = 6, P < 0.0005), indicating that extraction
yielded wax from a distinct layer inside the cuticle. Respec-
tive wax samples must therefore be interpreted as intracutic-
ular wax. The overall wax yield of all steps in this
experiment (epicuticular plus intracuticular wax) closely
matched the yield found for direct extraction of the same
surface in the previous experiment (ANOVA, N = 6,
P = 0.160).
The intracuticular and epicuticular wax layers shared
very similar qualitative and quantitative compositions,
both in terms of compound classes and chain length pat-
terns within fractions (Tables 1 and 2, Figs. 5 and 6). As
80
70
60
50
40
30
20
10
0
Epicuticular
Intracuticular
Total abaxial
Fig. 5. Relative amounts of wax compound classes within the epicuticular, intracuticular, and total abaxial wax mixtures on the second leaf of rye. The
results from the four gum arabic treatments were taken together to show the composition of the epicuticular waxes, whereas the final extraction results
represent the composition of the intracuticular waxes.

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X. Ji, R. Jetter / Phytochemistry 69 (2008) 1197–1207
two exceptions to this overall homogeneous wax composi-
tion, primary alcohols were found at higher percentage in
the epicuticular wax than in the intracuticular layer, while
steroids showed the opposite distribution. The latter result
confirmed previous findings for other species, where cyclic
components also established gradients going from higher
100
100
Epicuticular
B
A
Intracuticular
80
80
60
40
20
0
Total abaxial
60
40
20
0
24
26
28
30
24
26
28
30
Chain length
Chain length
100
80
60
40
20
0
D
C
30
20
10
0
26
28
30
32
27
28
29
30
31
32
33
Chain length
Chain length
Fig. 6. Chain length distributions of individual wax components in the epicuticular, intracuticular, and total abaxial wax mixtures on the second leaf of
rye. Percentages of individual homologs within the series of (A) fatty acids, (B) primary alcohols, (C) aldehydes and (D) alkanes are shown as means
(n = 6) with SD.
50
Epicuticular
Intracuticular
Total abaxial
40
30
20
10
0
19
21
23
25
27
Alkyl chain length
Fig. 7. Chain length distributions of alkylresorcinols in the epicuticular, intracuticular, and total abaxial wax mixtures on the second leaf of rye.
Percentages of individual homologs within the fraction are shown as means (n = 6) with SD.

X. Ji, R. Jetter / Phytochemistry 69 (2008) 1197–1207
1205
concentrations inside the cuticle to lower concentrations at
4.2. Wax extraction
the surface (Wen et al., 2006).
Finally, the most drastic gradient between the epicuticu-
lar and intracuticular waxes of rye leaves was found for the
ARs. These compounds could not be detected in the epicu-
ticular wax, but were instead restricted entirely to the
intracuticular layer of wax (Fig. 5). This layer was found
to contain 0.2 0.1 lg/cm2 of ARs, accounting for
2.0 0.8% of the intracuticular wax mixture. The AR frac-
tion was dominated by the homolog with C23 alkyl side
chain (Fig. 7). Overall, the concentration and chain length
patterns of ARs in the intracuticular wax samples largely
confirmed our previous findings for the overall wax mix-
ture from the abaxial side of the rye leaf. This result further
underlines that all the ARs present in the rye leaf cuticle are
largely restricted to the inner wax layer. They reach their
highest concentration slightly underneath the surface and
may be exposed at the true surface only in very small
amounts due to wax diffusion.
The total wax mixture was extracted by immersing the
whole leaf segments twice for 30 s into CHCl3 containing
n-tetracosane as an internal standard. The resulting solu-
tions were concentrated, transferred into small vials, taken
to dryness under a gentle stream of N2, and stored at 4 °C.
For selective extraction of either adaxial or abaxial wax,
respective sides of the rye leaf segments were brushed
approximately 60 times with fabric glass soaked with
CHCl3. Before use, the fabric glass was cleaned by thor-
ough extraction in CHCl3 using a Soxhlet apparatus. Tetr-
acosane was added as an internal standard and the wax
extracts were stored at 4 °C.
In order to discriminate between epicuticular and
intracuticular waxes, gum arabic was used as an adhesive
for mechanical removal of the outer layer and then solvent
extraction was employed to sample the inner wax layer.
Prior to use, gum arabic powder (Sigma–Aldrich) was thor-
oughly delipidated with CHCl3 in a Soxhlet apparatus,
dried and dissolved in distilled water (1 g mlÀ1). The glue
solution was spread onto the abaxial side of the leaf seg-
ment and air-dried for 40 min. The hardened adhesive film
was stripped off, broken into pieces and extracted with
CHCl3 containing tetracosane as internal standard. The
process was repeated three more times on the same side
of the leaf segment and the resulting wax solutions were
dried and stored at 4 °C. After the adhesive treatments,
the same side of the leaf segment was extracted by brushing
20 times with CHCl3-soaked fabric glass to sample the
intracuticular wax.
3. Conclusions
In summary, we have detected ARs with alkyl chain
lengths from C19 to C27 in the leaf cuticular waxes of rye.
The rye leaf ARs were found to be largely restricted to the
cuticle, with relatively low concentrations detected in the
internal lipids of this organ. ARs accumulated in similar
amounts in the cuticular waxes of both sides of the leaves.
While they were found at relatively high concentrations in
the intracuticular layers, they were absent from the epicuticu-
lar wax. This finding implies that the rye leaf ARs are located
very close to the surfaces of the leaves, but are covered by a
thin layer of epicuticular wax. It can be concluded that the
major AR amounts are not exposed at the very surface of
the tissue, and are not available for direct contact with micro-
organisms and insects that land on the leaf surface.
4.3. 5-n-Tridecylresorcinol (6, AR13:0)
Synthesis of AR13:0 (6) was carried out as described by
Furstner and Seidel (1997).
¨
4.4. 5-n-Nonadecylresorcinol (1, AR19:0)
4. Experimental
AR19:0 (1) was synthesized using similar methods as
described by Furstner and Seidel (1997): a solution of triflic
¨
4.1. Plant growth
anhydride (2.1 g, 7.4 mmol, Sigma–Aldrich) in CH2Cl2
(9 ml) was slowly added to a solution of 3,5-dimethoxyphe-
nol (7) (1.5 g, 9.8 mmol, Sigma–Aldrich) and 2,6-lutidine
(1.6 ml, 13.7 mmol, Sigma–Aldrich) in CH2Cl2 (48 ml) at
À10 °C. The solution was brought to 0 °C and stirred for
2 h before adding H2O (5 ml). The organic layer was
removed and dried with Na2SO4. After removing the sol-
vent under vacuum, the crude product was purified by flash
CC on packed silica gel with hexane:CH2Cl2 (1:1). After
removing the solvents and drying overnight, 3,5-dimeth-
oxyphenol triflate (8) was obtained as a yellow syrup with
90% yield. 1-tridecene (9) (474 mg, 0.7 ml, 2.6 mmol,
Sigma–Aldrich) and 9-borabicyclo[3.3.1]nonane (9-BBN)
(5.2 ml, 2.6 mmol, Sigma–Aldrich) in THF (60 ml) were
stirred for 2 h under N2 at room temperature. Then
NaOMe (0.17 g, 3 mmol), triflate (8) (0.65 g, 2.3 mmol)
Rye (Secale cereale L. cv. Esprit) seeds were purchased
from Capers, Vancouver and germinated directly in soil.
Plants were grown in several batches in a greenhouse at
the University of British Columbia (23 °C, 14 h light at
approximately 120 lmol mÀ2 sÀ1). Before the third leaves
had reached approximately half of their final length (typi-
cally three weeks after planting), the second leaves were
harvested and prepared for wax analysis. To this end, a
5 cm-long segment was cut out of the second leaf 10 cm
away from the point of emergence from the sheath of the
first leaf. Wax was sampled from the segment using one
of three methods (see below), and six independent parallels
were analyzed for each method. Sampled surface areas
were calculated based on leaf widths and segment length.

1206
X. Ji, R. Jetter / Phytochemistry 69 (2008) 1197–1207
and PdCl2 (dppf) (56 mg, 0.07 mmol, Sigma–Aldrich) were
added, the mixture was heated until reflux began, this being
maintained for 1 h, the solvent was next removed and
CH2Cl2 (10 ml) was added. Insoluble residues were
removed by passage of the CH2Cl2 solution through a
short column of silica. After evaporation of the solvent,
the crude product was purified by flash CC with hexane/
EtOAc (15:1) as eluent, to produce 1,3-dimethoxy-5-non-
adecylbenzene (10) as a colorless solid (54% yield). A mix-
ture of 10 (0.5 g, 1.2 mmol) and 9-iodo-9-BBN (0.41 ml,
2.52 mmol, Sigma–Aldrich) in hexane (25 ml) was stirred
for 3 h at room temperature. Then the solvent was removed
under vacuum and the residue dissolved in Et2O (15 ml).
Ethanolamine (0.14 ml, 2.2 mmol, Sigma–Aldrich) in
THF (1 ml) was added to precipitate the 9-BBN ethanol-
amine adduct, the mixture was stirred for 3 h, and the pre-
cipitate was filtered off. The filtrate was taken to dryness,
and the crude product was purified by flash column chro-
matography using hexane/EtOAc (2:1) as eluent, giving
analytically pure 1 (61% yield).
2 ml minÀ1. Individual compounds were quantified against
the internal standard by automatically integrating peak
areas. All quantitative data are given as means of six par-
allel experiments and standard deviations. Statistical anal-
yses were performed with SPSS 13.0 (SPSS, USA).
Acknowledgements
This work has been supported by the Natural Sciences
and Engineering Research Council (Canada), the Canada
Research Chairs Program, and the Canadian Foundation
for Innovation.
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