n 5? New ^ Phytologist 
Chemical composition of the epicuticular and 
intracuticular wax layers on the adaxial side of 
Ligustrum vulg?re leaves 
Christopher Buschhaus^ Hubert Herz^ and Reinhard Jetter^'^ 
^Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada; ^Smithsonian Tropical Research 
Institute, PO Box 0843-03092, Balboa, Anc?n, Republic of Panam?; ^^Department of Chemistry, University of British Columbia, 6174 University Boulevard, 
Vancouver, BC V6T 1Z3, Canada 
Author for correspondence: 
Reinhard Jetter 
Tel: +7 604 822 2477 
Fax: +7 604 822 6089 
Email: jetter@interchange. ubc. ca 
Received: 20 April 2007 
Accepted: 5 June 2007 
Summary 
? Previous research has shown that cuticular triterpenoids are exclusively found in 
the intracuticular wax layer of Prunus laurocerasus. To investigate whether this 
partitioning was species-specific, the intra- and epicuticular waxes were identified 
and quantified for the glossy leaves of Ligustrum vulg?re, an unrelated shrub with 
similar wax morphology. 
? Epicuticular wax was mechanically stripped from the adaxial leaf surface using the 
adhesive gum arable. Subsequently, the organic solvent chloroform was used to 
extract the intracuticular wax from within the cutin matrix. The isolated waxes were 
quantified using gas chromatography with flame ionization detection and identified 
by mass spectrometry. The results were visually confirmed by scanning electron 
microscopy. 
? The outer wax layer consisted entirely of homologous series of very-long-chain 
aliphatic compound classes. By contrast, the inner wax layer was dominated (80%) 
by two cyclic triterpenoids, ursolic and oleanolic acid. 
? The accumulation of triterpenoids in the intracuticular leaf wax of a second, unre- 
lated species suggests that this localization may be a more general phenomenon in 
smooth cuticles lacking epicuticular wax crystals. The mechanism and possible eco- 
logical or physiological reasons for this separation are currently being investigated. 
Keywords: cuticular wax, leaf surface, plant cuticles, privet {Ligustrum vulg?re), 
triterpenoids. 
New Phytologist (2007) 176: 311-316 
? The Authors (2007). Journal compilation ? New Phytologist (2007) 
doi: 10.1111/J.1469-8137.2007.02190.X 
Introduction 
The nonwoody surfaces of plants are separated from the 
surrounding atmosphere by the cuticle, a layer consisting 
of cutin and wax Qetter et ai, 2006). This wax is both 
impregnated in (intracuticular) and exterior to (epicuticular) 
the cutin biopolymer (Jeffree, 1986). Epicuticular wax may 
exist as a smooth film in some species, typically rendering 
their surfaces glossy, or it may be textured by protruding wax 
crystals in other species (Jeffree, 2006). The wax forms a 
barrier to deleterious water loss, which is the primary function 
of the cuticle (Baur, 1998). Other secondary functions have 
been suggested, such as ultraviolet (UV) light reflection 
(Reicosky & Hanover, 1978). Also, as the epicuticular wax is 
the first physical barrier encountered by external organisms, 
the wax likely plays a role in plant?insect (M?ller, 2006) and 
plant?pathogen interactions (Carver & Gurr, 2006). 
Plant cuticular waxes represent complex mixtures of 
very-long-chain aliphatics and cyclic compounds (Jetter et ai, 
2006). The aliphatics include fatty acids, aldehydes, primary 
www.newphytologist.org 311 
312    Research 
New 
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and. secondary alcohols, ketones, and alkanes, with chain lengths 
ranging from C^Q to C,g in homologous series. Alkyl esters 
from Cog to CjQ may also be present. In addition to these 
straight-chain compounds, cyclic compounds such as triter- 
penoids, tocopherols, and aromatic compounds may be found 
in either large or small quantities, depending on the species. 
The cuticular waxes of plants have traditionally been con- 
sidered to be a homogenous mixture, mainly because organic 
solvents indiscriminately extract both epi- and intracuticular 
"wax (Jetter et al., 2000). However, recently developed 
methods for physically removing the epicuticular wax before 
extraction with solvents permit the composition of each layer 
to be analyzed independently (Jetter et al., 2000; Jetter & 
Sch?flfer, 2001). Compounds present in the outer or inner 
"wax region of different plant species can now be identified. 
Initial studies on the glossy-leafed Prunus laurocerasus lepoited 
that the epicuticular wax was composed exclusively of 
aliphatic compounds, while the intracuticular "wax contained 
high percentages of two cyclic triterpenoids (Jetter etal, 2000). 
Triterpenoids are also known to form surface wax crystals on 
other species such as Ricinus communis (Guhling et al., 2006) 
and Macarangasp-p. (Markst?dter et aL, 2000). However, for 
glossy-leafed plants, it remains unkno"wn whether or not the 
localization of triterpenoids in the intracuticular wax of 
P. laurocerasus (Rosaceae) is a species-specific finding. To 
further explore this issue, the following questions "were 
addressed for the unrelated evergreen shrub Ligustrum vulg?re 
(Oleaceae) which also has glossy leaves devoid of epicuticular 
"wax crystals. (1) What is the quantity and composition of the 
cuticular "wax? (2) Is this composition homogenous? (3) If not, 
how does the epicuticular wax film differ from the intracuticular 
"wax? (4) How much does each layer contribute to the overall 
Materials and Methods 
Plant material 
Branches of Ligustrum vulg?re L. (privet) were collected from 
cultivated plants gro"wing on the campus of the University of 
Wuerzburg, Wuerzburg, Germany. Approximately 10 mature 
leaves were excised from the branch using a razor blade and 
pooled for each treatment. Five independent replicates "were 
analyzed per treatment. 
Mechanical wax removal 
Gum arable was employed as an adhesive for the selective removal 
of epicuticular waxes. Before the experiment, commercial gum 
arable powder (Sigma-Aldrich, OakviUe, Canada) was extracted 
in a Soxhlet apparatus with hot chloroform to remove any 
soluble lipids and residues. An aqueous solution of the 
adhesive (1 g ml~ ) was applied onto the entire adaxial surface 
of the leaves using a small paintbrush. After 30 min, the 
solution was dry and a thin polymer film could be peeled off 
in pieces "which were collected and extracted overnight with 
chloroform at room temperature. A defined amount of n- 
tetracosane "was added to the extracts as an internal standard. 
The treated surface area was subsequently measured digitally 
by scanning photocopies of the leaves. 
Wax extraction 
Total wax extraction from the adaxial surface was achieved by 
placing the intact leaf onto a flexible rubber mat, gently 
pressing a glass cylinder, 10 mm in diameter, onto the center 
of the exposed leaf surface and filling the cylinder with 
approx. 1.5 ml of chloroform (Jetter et al, 2000). The solvent 
was agitated for 30 s (by pumping "with a Pasteur pipette) and 
removed. When any solvent leaked between cylinder and leaf 
surface, the sample was discarded. Tetracosane was immediately 
added to all the extracts of cuticular waxes as an internal standard 
and the solvent "was removed under reduced pressure. A similar 
procedure was used to extract intracuticular waxes after 
mechanical removal of epicuticular "waxes from adaxial surfaces. 
Chemical analysis 
Before gas chromatography (GC) analysis, chloroform was 
evaporated from the samples under a gentle stream of 
nitrogen (N^) while heating to 50?C. Then the wax mixtures 
were treated with bis-A'i7V-(trimethylsilyl)trifluoroacetamide 
(BSTFA; Sigma-Aldrich) in pyridine (30 min at 70?C) to 
transform all hydroxyl-containing compounds into the 
corresponding trimethylsilyl (TMSi) derivatives. The qualitative 
composition was studied with capillary GC (5890N; Agilent, 
Avondale, PA, USA; column 30 m HP-1, 0.32 mm inner 
diameter, film thickness = 0.1 [am; Agilent) with helium 
(He) carrier gas inlet pressure programmed for constant flo"w 
of 1.4mlmin~' and mass spectrometric detector (5973N; 
Agilent). GC "was carried out with temperature-programmed 
injection at 50?C, with the oven temperature held for 2 min 
at 50?C, raised by 40?C min"' to 200?C, held for 2 min at 
200?C, raised by 3?C min'^ to 320?C and held for 30 min 
at 320?C. Individual wax components were identified by 
comparison of their mass spectra with those of authentic 
standards and literature data. The quantitative composition of 
the mixtures was studied using capillary GC "with flame 
ionization detector under the same GC conditions as above, 
but with hydrogen (H^) carrier gas inlet pressure regulated 
for a constant flow of 2 ml min~ . Single compounds were 
quantified against the internal standard by automatically 
integrating peak areas. 
Scanning electronic microscopy 
Samples consisting of untreated leaves and treated leaves (as 
described in the previous sections) were mounted on stubs 
New Phytologist (2007) 176: 311 -316 WWW.newphytologist.org    ?The Authors (2007). Journal compilation ? New Phytologist (2007) 
New 
Phytologist Research    313 
>>   15 
X (S 5    10 
c c c C O 0) (D o 
F E E b 
ns (6 (D 2 !? U 
0) <D 0) CO 
c > > > (/) (/) (0 
<D 0) Q 
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c o 
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F t3 to 
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'-        5        m 
Fig. 1 Wax yields from adaxial sides of Ligustrum vulg?re leaves 
sampled using a combination of mechanical and extractive methods. 
In a first experiment, the total cuticular wax was sampled directly 
using chloroform. In an independent second experiment, three 
consecutive treatments with the adhesive gum arable were employed 
to remove the epicuticular wax layer, followed by extraction with 
chloroform to remove the intracuticular wax layer Wax yields are 
given as mean values (? standard deviation; n = 5). 
using double-sided adhesive tape and allowed to air-dry 
overnight. Samples were then coated with 5 nm of gold (Au) 
in a Cressington Sputter Coater 208 H (Ted Pella, Inc., 
Redding, CA, USA) before viewing under a Hitachi S4700 
field emission scanning electron microscope (SEM; Nissei 
Sangyo America, Ltd, Pleasanton, CA, USA) at a 1-kV 
accelerating voltage and a 12-mm working distance. The 
order of magnitude of epicuticular film thickness was 
approximated from the height difference between adjacent 
untreated and gum arabic-treated surfaces. 
Results 
The cuticular wax components of the adaxial side of privet 
leaves were identified and quantified by GC and mass 
spectrometry. Preliminary experiments showed that two 
applications of room-temperature chloroform for 30-s 
durations exhaustively extracted the total cuticular "wax from 
privet leaves. With the chloroform restricted to the adaxial 
surface by a glass cylinder, these selective extractions produced 
a total of 28 + 3 (mean + standard deviation) (ag cm~ of wax 
from the adaxial surface (Fig. 1). The wax mixture contained 
six identifiable compound classes (Fig. 2). Triterpenoids were 
most abundant (66%), and were accompanied by smaller 
amounts of ?-alkanes (11 %), fatty acids (4%), primary alcohols 
(3%), branched alkanes (1%), and aldehydes (< 1%). For the 
total wax mixture, 13% could not be identified. 
25 
20 
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ax
 
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to <D 
m Q. 
Fig. 2 Composition by compound class of the total wax mixture 
(black bars) and the epicuticular (white bars) and intracuticular (gray 
bars) wax layers on the adaxial leaf surfaces of Ligustrum vulg?re. 
Amounts of the different compound classes are given as mean values 
(? standard deviation; n = 5). 
The triterpenoid class consisted of only two compounds, 
ursolic and oleanolic acid (Fig. 3). The remaining very- 
long-chain compound classes were present as homologous 
series. The ?-alkanes ranged from Cjj to C,j, with tritriacon- 
tane (C,,) as the dominant homolog. Similarly, the ?yo-alkanes 
were also dominated by odd-numbered carbon numbers (Cjn 
and Cjj), but with even-numbered chain lengths (Cjg and 
C,Q, respectively). Conversely, anteiso-a??anes had even carbon 
numbers (Coj and C,^), as did the other compound classes. 
Fatty acids and primary alcohols ranged from C2Q and Cjj to 
C,^, respectively, peaking at Cjj. Tetratriacontanal (C,^) was 
the only aldehyde detected. 
Scanning electron microscopy revealed that the adaxial sur- 
face of privet leaves contained only irregularly margined, flat, 
epidermal pavement cells (no guard cells or trichomes; data 
not shown). The surface was minutely granulated but lacked 
distinguishable surface crystals (Fig. 4a). Overall, the surface 
appeared to be covered by a relatively smooth film of wax. A 
single application of gum arable produced a very smooth sur- 
face with a curvilinear line dividing the leaf between native 
and gum arabic-treated regions. Although a single application 
of gum arable removed most of the epicuticular wax, small 
patches of epicuticular wax remained. However, three consecu- 
tive applications of gum arable essentially removed all of the 
epicuticular wax film (Fig. 4b). 
Preliminary experiments showed that the adhesive gum 
arable reproducibly removed the surface wax from privet 
leaves (data not show^n). When the adaxial surface was treated 
repeatedly, the wax yields decreased and were approaching 
) The Authors (2007). Journal compilation ? New Phytologist (2007)     WWW.newphytologist.org New Phytologist (2007) 176: 311-316 
314    Research 
New 
Phytologist 
20 
15 
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Fig. 3 Composition of (a) the total wax 
mixture and the (b) epicuticular and (c) 
intracuticular wax layers on adaxial leaf 
surfaces of Ligustrum vulg?re. Compounds 
are listed according to chain lengths in the 
homologous series of aliphatic compound 
classes, or as triterpenoid isomers. Relative 
quantities of the individual compounds are 
given as mean values (? standard deviation; 
n = 5). 
zero after the third adhesive application (Fig. 1). The wax yield 
of three consecutive gum arable treatments on the adaxial leaf 
surface of privet added to 6.6 + 1.6 jag cm . This epicuticular 
"wax consisted of alkanes (55%), branched alkanes (5%), fatty 
acids (18%), primary alcohols (8%), and aldehydes (< 1%; 
Fig. 2). Notably absent were the triterpenoids. The chain 
length distributions matched those found in the total wax 
mixture (see above). Hentriacontane (C,^) and tritriacontane 
(C,,) were the most abundant compounds and were present 
at nearly equivalent amounts (Fig. 3). Together they com- 
prised nearly 36% of the epicuticular wax. 
Solvent extraction of the adaxial leaf surface after the gum 
arable treatments yielded 25.6 + 8.3 jig cm~^ (Fig. 1). The 
intracuticular wax was strongly dominated by triterpenoids 
(80%). Small quantities of alkanes (3%), fatty acids (1%), and 
alcohols (1%) were also present (Fig. 2). Branched alkanes 
and aldehydes were not detectable. The aliphatics tended to 
have shorter chain lengths than in the total cuticular wax, 
with maxima for hentriacontane (C,j), hexacosanoic acid 
(Cjg), and octacosanol (C^g; Fig. 3). The quantities of the two 
triterpenoids relative to each other resembled those found in 
the total cuticular wax. 
Discussion 
The compounds identified and quantified in the adaxial wax 
of L. vulg?re leaves have all been reported in plant waxes 
before. The very-long-chain aliphatics were typical of the 
general suite of straight chain compound classes and chain 
lengths found in the waxes of many other species. Also, the 
New Phytologist (2007) 176: 311 -316 WWW.newphytologist.org    ?The Authors (2007). Journal compilation ? New Phytologist (2007) 
New 
Phytologist Research    315 
(a) (b) 
? 
te         mm _   ^ 
Fig. 4 Scanning electron micrographs of Ligustrum vulg?re adaxial 
leaf surfaces before and after treatment with gum arable, (a) The 
adaxial surface after a single treatment with gum arable (top right) is 
clearly delineated from the epicuticular wax film-covered native 
surface (bottom left), (b) Three consecutive treatments with gum 
arable produce a smooth adaxial surface. Bars, 1 pm. 
two triterpenoids present were the same as those found in 
7? laurocerasus (Jetter et aL, 2000). 
Although the above analysis of the total soluble lipids 
allows the identification of all compounds present, it does not 
reveal possible partitioning of compounds within the cuticle. 
Is the "wax composition homogenous for privet leaves or do 
compounds tend to partition along an inside-to-outside 
gradient? To answer this question, the epicuticular wax film 
overlying the adaxial surface was analyzed separately from the 
intracuticular wax. 
Repeated application of gum arable to the adaxial surface 
of privet leaves resulted in continually decreasing yields. This 
demonstrated the selective removal by gum arable of surface 
wax up to the mechanically resistant cutin matrix. However, 
the subsequent extraction Wth chloroform yielded a nearly 
50-fold increase in extracted wax as compared w^ith the third 
gum arable treatment. Thus, the mechanically removed wax 
must be interpreted as epicuticular wax while the remaining 
wax subsequently extracted with solvent must be intracuticular 
wax. Moreover, because the combined yields of epicuticular 
plus intracuticular wax (sum: 32 + 9 }ag cm~ ) were confirmed 
by a second independent experiment using total wax extraction 
(28 + 3 }ag cm" ), the selective extractions of epi- and intra- 
cuticular wax were also exhaustive. These results for the total 
wax loads on the adaxial sides of L. vulg?re leaves also match 
the wax quantity found on the adaxial side of P. laurocerasus 
leaves (28 jag cm~^; Jetter et ai, 2000). 
Scanning electron microscopy images visibly supported 
these chemical data. Only small patches of epicuticular wax 
were remaining after a single application of gum arable, and 
were no longer visible after three consecutive applications. 
These findings parallel the diminishing quantities of wax 
found by chemical analysis of consecutive gum arable 
applications. Using the quantity of mechanically removed 
wax (6.6 + 1.6 (ag cm" )  and an approximate density of 
0.8-1.0 X lO'' g m"^ for very-long-chain aliphatics (Le Roux, 
1969), the epicuticular film thickness was expected to be 
65 ? 80 nm. This matches the magnitude of film thickness 
observed by SEM. 
The relative partitioning of wax compounds on the adaxial 
side of privet leaves can now be accurately described. The 
identified compounds in the outer layer were exclusively 
very-long-chain aliphatics, mth the majority of compounds 
being alkanes. No triterpenoids were detectable. In contrast, 
the intracuticular wax was composed almost entirely of two 
triterpenoids, ursolic acid and its isomer oleanolic acid, which 
were present in a 3 : 1 ratio, respectively. Less than 5% of this 
inner wax layer was aliphatic in nature. This quantity of 
aliphatics is within the error margin of complete epicuticular 
wax extraction. 
The aliphatic constituents of the epi- and intracuticular 
waxes differed in their relative amounts. Branched alkanes 
and aldehydes were detectable only in the epicuticular wax, 
although even in the outer layer these were present at very low 
quantities. More strikingly, the intracuticular aliphatics all 
contained shorter chain lengths for compound class maxima 
and shorter homolog ranges compared with the epicuticular 
wax. Because compounds with shorter chain lengths are 
slightly more polar than longer compounds mthin the same 
compound class, the intracuticular aliphatics contained a 
higher proportion of polar constituents. This same pattern of 
shorter intracuticular and longer epicuticular aliphatic com- 
pounds has been found in the leaf and needle waxes of Rubus 
fruticosus and Taxus baccata, respectively (Haas & Rentschler, 
1984; Wen et aL, 2006). The present work further supports 
the hypothesis that a gradient occurs between outer unpolar 
and inner less unpolar wax. 
Prunus laurocerasus cuticles, which have the same two triter- 
penoids as privet, displayed the same intracuticular partitioning 
of the triterpenoids. Also, similar differences between epi- and 
intracuticular wax, albeit with different triterpenoids, 
have been reported in other species, including the leaves 
oiMacaranga tanarius (Guhling et al, 2005), the stems of 
Ricinus communis (Guhling et aL, 2006) and the fruit of 
Lycopersicon esculentum (Vogg et aL, 2004). Although some 
triterpenoids have been show^n to produce surface crystals, no 
cases have been reported where triterpenoids are present in a 
smooth epicuticular wax film. This clearly suggests that, in 
general, for species with glossy cuticles, the triterpenoids tend 
to be located exclusively in the intracuticular wax. 
The mechanism for establishing such a gradient remains to 
be determined. It is possible that the physicochemical properties 
of triterpenoids, including the size and polarity of the mole- 
cules, may hinder their outward movement. Alternatively, 
they may interact with other intracuticular components such 
as cutin or cellulose microfibr?s that extend from the epidermal 
cell wall. Moreover, it has been suggested that physiological or 
ecological functions may be linked to such partitioning (Jetter 
et aL, 2000), but they also remain to be investigated. 
) The Authors (2007). Journal compilation ? New Phytolo^st (2007)    WWW.newphytologist.org New Phytologist (2007) 176: 311-316 
316    Research 
New 
Phytologist 
In conclusion, the identified composition of the outer 
adaxiai surface film on privet leaves was comprised entirely of 
very-long-chain aliphatics. Conversely, the vast majority of 
the intracuticular wax consisted of two triterpenoids. This 
very closely matches the transversal gradients of compounds 
in the leaf cuticle of the unrelated species P. laurocerasus, and 
thus suggests that the pattern may be generalized across many 
species that contain triterpenoids in smooth plant cuticles. 
Acknowledgements 
The authors would like to acknowledge technical help by 
Stephanie Full, Stephan Knapek, Lisa Brumm, and the 
University of British Columbia Biolmaging Facility staff. This 
work has been supported by the Deutsche Forschungsgesellschaft 
(Germany), the Natural Sciences and Engineering Research 
Council (Canada), the Canadian Research Chairs Program, 
and the Canadian Foundation for Innovation. 
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