Epicuticular Waxes
PJ Holloway, The Long Ashton Research Station, Bristol, UK
CE Jeffree, University of Edinburgh, Edinburgh, UK
Ó 2017 Elsevier Ltd. All rights reserved.
This article is a reproduction of the previous edition article by P.J. Holloway, C.E. Jeffree, volume 3, pp. 1190–1204, Ó 2003, Elsevier Ltd.
Nomenclature
Functional groups Unbranched ¼ n-(normal);
branched ¼ br (eCH(CH3)e), e.g., iso-(2-methyl), anteiso-
(3-methyl), etc.; alkane (eCH3) ¼ –ane; unsaturation
(eHC]CHe) ¼ –ene; alcohol (eOH) ¼ -ol or hydroxy-;
ketone (eCOe) ¼ -one or oxo-; aldehyde (eCHO) ¼ -al;
acid (eCOOH) ¼ -oic acid; ester (eCOeOe) ¼ -yl for
alcohol moiety then -oate for acid moiety
Definitions
In the generic sense, the terms wax or waxy are used to describe
the physical characteristics of certain classes or mixtures of
organic substances. Such materials are inert solids or semisolids
with melting points in the range 35–100  C. They are insoluble
in water but soluble in apolar solvents, such as chloroform,
benzene, and diethyl ether. In addition, most waxes will form
hard, glossy films on polishing; these coatings have much lower
free surface energies than glass, for example, and are not easily
wetted by water.
A wide variety of natural products fitting this general
description can be obtained from plant, animal, and mineral
sources, although nowadays many commercial waxes are man-
ufactured synthetically. However, waxes cannot be defined
unequivocally in simple chemical terms because they often
differ markedly from one another in composition. Neverthe-
less, many natural waxes consist of complex mixtures of long-
chain nonpolar compounds, with the molecular weights of
individual constituents commonly ranging from about 200 to
600, but occasionally exceeding 1000; these values correspond
with chain lengths of between about 20 and 60 carbon atoms.
Occurrence
Waxes are omnipresent in extant higher plant species, as well as
mosses and ferns, implying that these compounds appeared early
in the evolution of land plants, despite the fact that they are poorly
preserved in the macrofossil record. Most plant waxes are found in
intimate association with the cuticle, the thin continuous
nonliving ‘skin’ that covers the aerial surfaces of all plants. These
cuticular waxes are synthesized exclusively by the underlying
epidermal cells (see Secondary Products: Wax Pathways), as are
the unique series of C16 and C18 hydroxy and epoxy alkanoic
acids, which interesterify to form the characteristic polyester cutin
matrices of cuticular membranes themselves. Wax constituents
are subsequently secreted not only onto the surface of the cuticle
but also become embedded within it. The former are usually
referred to as ‘epicuticular’ or ‘superficial,’ but may sometimes
374 Encyclopedia of Applied Plant Sciences, 2nd edition, Volume 2
Primary carbon number and systematic stem name 10,
decan-; 20, icosan-; 30, triacontan-;40, tetracontan-
Prefixes for intermediate homologs þ1 ¼ hen-, þ
2 ¼ do-, þ 3 ¼ tri-, þ 4 ¼ tetra-, þ 5 ¼ penta-, þ
6 ¼ hexa-, þ 7 ¼ hepta-, þ8 ¼ octa-, and þ9 ¼ nona-
be called ‘surface lipids.’ ‘Intracuticular’ is used to denote waxes
present within the cuticle, but these will not be dealt with in
this article. The primary physiological functions of the cuticle
complex are to waterproof (liquid water) the plant surface and
to limit water loss (water vapor) from internal tissues.
Isolation and Analysis
The extraction of epicuticular waxes from plant material is
straightforward and usually involves brief exposure of the
surfaces of detached intact organs, such as leaves and fruits,
to an appropriate organic solvent; chloroform is often the
solvent of choice. According to the level of refinement required
for the investigation, this may be achieved by:
l total immersion of individual plant parts for about 30 s in
a large volume of solvent;
l running a fine stream of solvent over the selected surfaces
and collecting the effluent;
l wiping selected regions of the surface with extractive-free
cotton wool swabs soaked in solvent, followed by further
extraction of the swabs in the same solvent.
In each case, solvent washings are combined, then evapo-
rated, and the solid residue obtained dried to constant weight.
Overexposure to solvents can cause leaching of intracuticular
waxes and, if severe, may result in removal of internal lipids
and pigments from the specimens.
Such extraction procedures are often combined with gravi-
metric or planimetric measurements of the plant samples in
order to obtain a quantitative assessment of the mean epicutic-
ular wax content per unit weight or area. The amount of wax
on leaf surfaces may be as little as 1 mg cm2 on some temperate
species, but can reach as much as several mg cm2 on a number
of tropical palm species; in the latter case, these wax layers are so
thick that they can be removed by mechanical methods. The wax
deposits on the fruits of a given species are invariably heavier
than those on the corresponding leaves, which in turn may carry
different amounts of wax on their adaxial and abaxial surfaces.
The effects of solvent washing on a plant surface can also be
http://dx.doi.org/10.1016/B978-0-12-394807-6.00075-7

Wax Extract
Prep TLC
Analytical TLC Derivatization
chromogenic agents
wax fractions
GC
GC-MS
Figure 1 Summary of analytical procedures most commonly used to
determine the qualitative and quantitative composition of plant epicutic-
ular waxes. Adsorption thin-layer chromatography (TLC) fractionates
a wax into its constitute classes according to their polarity (Tables 1
and 2). Individual compounds are separated from one another by gas–
liquid chromatography (GC) and identified by mass spectrometry (MS).
monitored by scanning electron microscopy (SEM) in order to
confirm the efficiency of removal of epicuticular deposits.
A summary of typical analytical procedures used in the
determination of the composition of epicuticular waxes is
given in Figure 1. Thin-layer chromatography (TLC) on silica
gel provides useful qualitative information about the various
classes of constituents present by comparisons of RF values
observed with those of appropriate reference compounds,
together with spray reagents for specific functional groups in
some cases. TLC is also valuable as a preparative method for
isolating fractions corresponding with individual wax classes;
these can then be examined in more detail by other chromato-
graphic or spectroscopic techniques.
Gas–liquid chromatography (GC) with flame ionization
detection (FID) is used to identify and quantify each constit-
uent in a wax, although preliminary derivatization of any
free hydroxyl or carboxylic acid groups present is normally
carried out; the former are usually analyzed as the correspond-
ing trimethylsilyl ethers, the latter as methyl esters. Stationary
phases capable of operating at temperatures well above 300  C
are necessary in order to achieve complete separations; short-
length, bonded, fused silica capillary columns in conjunction
with both temperature and gas flow programming are used
routinely nowadays. Because such columns also offer high
resolution, they can be used as a one-step method of analysis
for a total wax extract. Compounds are often characterized
first from their retention indices following coinjection with
a series of known n-alkanes, whereas quantitation is achieved
by subsequent addition of an appropriate internal standard
and calculation of response factors with reference to model
Secondary Products j Epicuticular Waxes 375
compounds for each class of constituent. Final identification
of compounds is made by mass spectrometry (MS) from their
molecular weights and diagnostic fragmentation patterns;
some examples are shown in Figures 2–5. The most powerful
analytical tool available to the wax chemist today is still GC
coupled to MS (GC-MS). However, if single constituents can
be isolated from a wax, confirmatory structural information
may be obtained by nuclear magnetic resonance (NMR).
High-performance liquid chromatography (HPLC) has found
only limited use for wax analysis, mainly due to the
difficulty in detecting eluted compounds.
Composition
The chemistry of the wax secreted onto the surfaces of plants
shows a great diversity in composition and complexity, with
major differences not only between species, but also sometimes
even between different regions on the same species. The adaxial
and abaxial surfaces of pea (Pisum sativum) leaves, and the
sheaths and blades of the flag leaves of barley (Hordeum vul-
gare), are striking examples of the latter. On the other hand,
the composition of waxes from disparate species can some-
times be remarkably similar, as in the case of the leaves of
cabbage (Brassica oleracea) and Clarkia elegans or of the leaves
of tulip (Tulipa gesneriana) and the needles of Sitka spruce
(Picea sitchensis). Nevertheless, wax biosynthesis and its ulti-
mate deposition on the cuticle surface are under strict genetic
regulation. This factor is highlighted by the existence of several
spontaneous or induced subglaucous or ‘waxless’ mutants of
a number of crop species (peas, brassicas, corn (Zea mays;
maize), sorghum (Sorghum bicolor), and barley) and recently
in Arabidopsis; these plants show drastic alterations in the
composition, amount, or microstructure of their foliar waxes,
compared with the normal form.
It is difficult to classify plant waxes chemically, but they can
be divided roughly into those that contain predominantly
long-chain aliphatic constituents and others that possess large
amounts of cyclic constituents, especially triterpenoids; inevi-
tably, intermediate types with both categories of compound
are also known. In exceptional cases, as on some ferns, the
‘wax’ or ‘farina’ found on the fronds is composed almost
entirely of crystalline lipophilic flavonoids.
Many aliphatic compounds have been identified in epicutic-
ular waxes and these belong to various classes, which, in turn,
invariably show either a predominantly odd or even carbon
number; some representative examples are given in Tables 1
and 2, respectively. Plant species differ from one another in
the numbers of these classes present and their relative amounts,
as well as in the homolog content within each class. Homologs
differ from one another by one eCH2e group. Positional isom-
erism is commonly encountered in classes that possess one or
more functional group, such as hydroxy and oxo, in midchain
positions. Methyl-branching at terminal positions may some-
times be detected in certain classes, especially alkanes and ester-
ified primary alcohols and alkanoic acids.
A comprehensive survey of wax chemistry is outside the
scope of this article, but it is worth recording here, for reference,
the main aliphatic constituents present in the leaf waxes of some
familiar plant species. High proportions of alkanes are found on
376 Secondary Products j Epicuticular Waxes

100 M-29
57
Alkanes M-29
393

CH3CH2 CH CH2R 10 anteiso -C30

CH3
43 M-57 M-57 M+
71

Relative intensity
365 422
0
85 M-43
50 M-15 393
CH3 CH CH2R 10 iso -C31
CH3 M-15
M-43 421 M+
99 M-15 436
0
127
155 183 211 n-C31 M+
239 436

40 100 200 300 400

m/e

Figure 2 Mass spectra of n-hentriacontane (n-C31), 2-methyltriacontane (iso-C31), and 3-methylnonacosane (anteiso-C30) obtained from a GC-MS
analysis of the alkane fraction isolated from the epicuticular wax of tomato fruits. Fragmentation schemes showing the diagnostic ions for the two
types of branched (br) homologs are also included; only the high ends of the spectra are shown, the lower ends being similar to that of the n-
homolog.

100 43 71
[+H]
170
57
155
O
||
CH3(CH2)17 H2C C CH2 (CH2)7CH3
Relative intensity

155
58 295
310
[+H]
50
171 295
85

170
96 110
310 M+
127 183 311 422
250

40 100 200 300 400

m/e

Figure 3 GC-MS of the C29 ketone fraction isolated from the epicuticular wax of plum (Prunus domestica) fruits. The fragmentation scheme for the
major component, n-10-nonacosane, is shown.

100
313
Relative intensity

327 313
CH3(CH2)15 CH (CH2)13CH3 CH3(CH2)14 CH (CH2)14CH3
OSiMe3 OSiMe3
50 299 313

73 299 327
75 M−15
57 M+
83 103 129
285 341 509
524
40 100 200 300 400 500
m/e

Figure 4 GC-MS of the C31 secondary alcohol fraction isolated from the epicuticular wax of pea leaves, after conversion to the corresponding
trimethylsilyl (–OSiMe3) derivatives. The fragmentation schemes for the major components, n-16-(trimethylsiloxy)hentriacontane and n-15-
(trimethylsiloxy)hentriacontane, are shown.
 Secondary Products j Epicuticular Waxes 377

100
203 + Methyl TMSioxyursolate
E
+ E
D E
262 D CO2Me

Relative intensity
C D CO2Me
A B
Me3SiO

50

73 190 +CH
189 2 M-15
55 95 133 A B
M+
279 Me3SiO 542
527

40 100 200 300 400 500

m/e

Figure 5 GC-MS of ursolic (3b-hydroxyurs-12-ene-28-oic) acid isolated from the epicuticular wax of apple leaves, after conversion to the
corresponding trimethylsilyl (–OSiMe3) ether methyl ester (–CO2Me) derivative. The origin of the diagnostic retro-Diels-Alder rearrangement ion (m/z
262) is shown, together with those of other important fragmentation ions.

Table 1 Principal classes of aliphatic constituents with exceptions, such as esters, the most common homologs in an
a predominantly odd carbon number found in plant odd-numbered class are generally C29, C31, and C33, compared
epicuticular waxes with C26, C28, and C30 for an even-numbered class. However,
attempts to utilize wax chemistry as a chemotaxonomic marker
Classa Common examples
have only met with limited success.
Alkanes n-Nonacosane Plant waxes that are rich in cyclic constituents are less
n-Hentriacontane common than their predominantly aliphatic counterparts.
Ketones n-15-Nonacosanone They are usually found coating the surfaces of fruits or isolated
n-16-Hentriacontanone from leaves that exhibit a glossy finish, such as Rhododendron
Secondary alcohols n-10-Nonacosanol species and cherry laurel (Prunus laurocerasus). Pentacyclic triter-
n-15-Nonacosanol penoids and sterols (tetracyclic triterpenoids) are the two main
b-Diketones n-14, 16-Hentriacontandione
classes of cyclic constituents that occur most frequently,
n-16, 18-Tritriacontandione
although the latter are more commonly detected as minor
a components. Pentacyclics with ursane and oleanane skeletons
Arranged in increasing order of polarity.
are the most abundant classes. Ursolic acid and its analogs
are the major constituents of apple (Malus pumila) fruit waxes,
Table 2 Principal classes of aliphatic constituents whereas oleanolic acid and its derivatives dominate in those
with a predominantly even carbon number found in from olives (Olea uropaea) and grapes (Vitis vinifera); a- and
plant epicuticular waxes b-amyrins, also of the oleanane series, can be detected in
many leaf waxes. The ubiquitous plant sterols, b-sitosterol,
Classa Common examples
campesterol, and stigmasterol, are often secreted onto the
Esters n-Octadecyl hexacosanoate surface of foliage.
n-Hexadecyl octacosanoate It should be noted that esterification is a widespread mech-
Aldehydes n-Hexacosanal anism for the formation of wax components. This may occur
n-Octacosanal with any aliphatic or cyclic constituent containing hydroxyl
Primary alcohols n-Hexacosanol or carboxylic acid groups. Some examples are esters of sterols
n-Octacosanol with alkanoic acids > C16 and triterpenols with acetic acid.
Acids n-Hexacosanoic
Very long-chain esters, known as estolides, are found in many
n-Octacosanoic
conifer waxes. These compounds are formed by the linear
a interesterification of various o-hydroxyalkanoic acids (e.g.,
Arranged in increasing order of polarity.
n-16-hydroxyhexadecanoic), a,u-alkanediols (e.g., n-hexade-
cane-1,16-diol), alkanoic acids and, sometimes, primary alco-
the leaves of peas and brassicas, aldehydes on sugar cane (Sac- hols; the corresponding molecular weights range from about
charum officinarum) leaves and lemon (Citrus limon) fruits, 800 up to 1500. Such compounds might provide the ‘missing’
ketones on leek (Allium porrum) leaves, primary alcohols on link between the cuticular lipids, wax, and cutin.
the leaves of many grasses and cereals, symmetric secondary
alcohols on the leaves of brassicas and C. elegans, asymmetric
secondary alcohols on some mosses, the needles of many Physical Form
gymnosperms and the leaves of carnations (Dianthus spp.),
and b-diketones on the leaves of many eucalypts, box (Buxus The fine structure of epicuticular wax layers in situ has been
sempervirens), and barley. Although there are some notable studied extensively over the last 35 years or so with the aid of
378 Secondary Products j Epicuticular Waxes
the scanning electron microscope (SEM), using both coated
and uncoated, fresh or dried specimens. Indeed, Barthlott
and his group have cataloged over 10 000 species and
proposed a classification scheme for use in the systematics of
angiosperm families. Epicuticular waxes show a great diversity
in ultrastructure, which can range from an extremely thin amor-
phous film to complex layers of microcrystals with different
shapes and sizes (0.1–20 mm). Common crystal types observed
are hollow tubes, simple or decorated platelets, rodlets, fila-
ments, and ribbons, which are generally flattened and exhibit
different degrees of adornment. Wax granules having an inde-
terminate shape may also be present; these may represent trun-
cated tubes, plates, or cuboid crystals. Although these different
crystal types often appear to be distributed randomly over the
cuticle surface, they can sometimes be found in regular arrange-
ments or definite patterns. For example, on Convallaria majalis
leaves, wax plates standing on their edges form parallel arrays
over large areas and also appear to radiate from the stomatal
apertures. On the other hand, platelets arranged in rosettes
appear to be a feature of the epicuticular wax layers in the order
Fabales. In the Strelitziacea, patterning extends to the forma-
tion of remarkable wax chimneys surrounding the stomatal
complexes. The walls of these tubes are constructed from fused
co-aligned wax crystals that appear to arise from a well-defined
ring of cuticle at the base. Such patterning effects indicate that
there is precise control over the development of wax
morphology in some species, but how this is achieved is not
known. If many wax crystallites occur on the cuticle surface,
they scatter short wavelength light and, collectively, are respon-
sible for the bluish matt ‘bloom’ visible on many leaves, stems,
and fruits. Cogent examples are the fruits of plums and grapes,
the leaves of brassicas, cereals, Eucalypts, carnations, and the
bloodroot (Sanguisorba canadensis), the latter celebrated by
M.C. Esher in his lithograph, ‘Dewdrop.’ Smooth glossy leaves,
like Rhododendron spp. and cherry laurel, are usually character-
ized by the presence of a dense amorphous covering of epicu-
ticular wax. Sometimes, the epicuticular wax layer may feel
greasy to the touch, as can be detected on some cultivars of
apple fruit.
Relationships between Composition and
Ultrastructure
Tube-like crystals of epicuticular wax occur widely in the plant
kingdom and chemical analyses have revealed the presence of
two main types of constituent in the corresponding isolated
waxes. The first group contains substantial amounts of asym-
metrical secondary alcohols, in which n-10-nonacosanol and
closely related positional isomers predominate; b-diketones
are the main components of the second type. Familiar exam-
ples of species containing these compounds have already
been cited above. The chemical distinctions between the two
types of tube are also supported by some morphological differ-
ences. Thus, those of the secondary alcohol class are usually
shorter and wider (0.3–1.1 mm long by 0.1–0.2 mm wide)
than those of the b-diketone class (2.0–3.0 mm long by 0.2–
0.3 mm wide).
Tubular crystals with morphologies different from those
described above are also known. For example, on leaves of
brassicas, C. elegans, and Centranthus ruber there are large
wax tubes (up to 8.0 mm long by 1.0 mm wide) that are trans-
versely ridged and taper gradually from base to apex. These
waxes all contain mixtures of alkanes and symmetrical
secondary alcohols and ketones. On the other hand, the
tubules that are found on the surface of Cerinthe minor leaves
are associated with the occurrence of d-lactones (1,5-
alkanolides) in the wax.
Other correlations between chemistry and ultrastructure
have also been made. Platelets are often formed on plant
surfaces by epicuticular waxes that contain high proportions
of primary alcohols (see above for examples). Similar associa-
tions between aldehydes and the formation of wax filaments,
and between symmetrical ketones and ridged rodlets, have
been observed on disparate groups of plant species. It should
also be noted that wax fine structure can differ markedly
according to the position on the same plant. There are b-dike-
tone-rich tubes on the abaxial surfaces of the flag leaves of
wheat (Triticum spp.) in contrast to primary alcohol-rich plates
on the corresponding adaxial surfaces, whereas, in barley, the
former predominate on the leaf sheaths and the latter on
both surfaces of the leaves.
Recrystallization experiments in vitro using volatile organic
solvents can be used to confirm possible crystal structure–
chemistry relationships for epicuticular waxes. Thus, total
waxes, individual class fractions isolated from them and, in
some cases, pure synthetic compounds can be compared in
a model system and monitored by SEM. However, the results
that have been obtained from such investigations fall into three
broad categories:
l Good agreement between the crystalline form of the
epicuticular wax in situ, that of the total wax after recrys-
tallization in vitro, and that of one of the components of the
wax after purification and recrystallization in vitro.
l Good agreement between the crystalline form of the
epicuticular wax in situ and that of the total wax after
recrystallization in vitro, but not that of any of the compo-
nents of the wax after purification and recrystallization
in vitro.
l No agreement between the crystalline form of the epicu-
ticular wax in situ and in vitro after recrystallization.
Examples in the first category include n-10-nonacosanol in
many monocotyledons, dicotyledons, and gymnosperms,
b-diketones and hydroxy-b-diketones in cereals and Eucalypts,
and n-hexacosanol and n-octacosanol in grasses and legumes.
Micrographs illustrating the morphology of epicuticular wax
layers and crystal forms in situ and in vivo are shown in
Figures 6–13.
Possible Mechanisms for Wax Secretion In Planta
The mechanism for transport of epicuticular wax constituents
from the epidermis, through the cuticle and onto the plant
surface has been the subject of much debate and experimen-
tation since the nineteenth century, but remains an enigma
to this day. For a long time, these materials were thought
to be extruded in a liquid or semisolid form via pores; this
hypothesis was based mainly on the visual interpretation
 Secondary Products j Epicuticular Waxes 379

(a) (a)

(b)

(b)

10-Nonacosanol

FID Response
Figure 6 Amorphous wax films. (a) The abaxial surface of Rhododen-
dron ponticum leaves (550) is covered with a dense crust of
triterpenoid-rich epicuticular wax, which thins out in the vicinity of the
numerous stomata present. (b) The adaxial surface of field bean (Vicia
faba) leaves (880) is finely corrugated, replicating the convex shape
Hentriacontane
of the underlying epidermal cells. The surface of the cuticle appears
smooth and has an extremely thin coating (c. 3 mg cm2) of wax.
5,10-Nonacosanediol

of the large cylindrical wax structures observed with the light

microscope on leaves such as those of Strelitzia, banana
(Musa spp.), bamboo (Arundinaria spp.), and the nodes of
sugar cane stems (Saccharum officinarum). However, succes- 370° 310° 250° 190° 130°
sive detailed investigations by both light and electron 40 30 20 10 0
microscopy have consistently failed to provide unequivocal RT (min)
evidence for the existence of either pores or any other form
of discrete channel through which waxes might be able to Figure 7 (a) The small tubular microcrystals of epicuticular wax found
migrate. The perceived problem for transport then is the on the adaxial surface of Aquilegia vulgaris leaves (5200) contain large
fact that wax molecules are large, involatile, and most will amounts of n-10-nonacosanol. (b) GC analysis of total wax after silyla-
be hard solids at physiological temperatures. Consequently, tion; Dexsil 300 column with flame ionization detection (FID).
it is difficult to envisage how they would be able to move
in their native state directly through the complex architecture
of either the cell wall or the cuticle. The in vitro experiments semicrystalline layer by self-association. To date, headspace
carried out with isolated waxes and their individual constit- analysis has not been able to detect the presence of volatile
uents suggest that a solvent or some other form of solubi- compounds specifically emitted from the surfaces of very
lizing agent might be instrumental in the transport process. glaucous plants and therefore potential candidates as wax
This could facilitate the permeation of wax in a liquid solvents. However, acyl carrier proteins have been identified
form through the cuticle. Once on the surface and exposed recently in the epicuticular waxes of broccoli (B. oleracea var.
to the atmosphere, the transport medium would then evap- italica); such proteins might be able to provide the missing
orate or dissociate, leaving the wax to form a crystalline or piece in the jigsaw puzzle.
380 Secondary Products j Epicuticular Waxes

(a) (a)

(b)
(b)

1-Hexacosanol
16,18-Tritriacontanediol

FID Response
FID response

1-Octacosanol

Hentriacontane

C31 diol
Monoesters
C29 diol C46 C42

370° 310° 250° 190° 130° 370° 310° 250° 190° 130°

40 30 20 10 0 40 30 20 10 0
RT (min) RT (min)

Figure 8 (a) The aggregations of long tubular microcrystals of epicu-
ticular wax that occur on the adaxial surface of Eucalyptus globulus
leaves (2200); they are characterized by the presence of n-16,18-
tritriacontanedione as the major component. (b) GC analysis of total
wax after reduction with sodium borohydride to the corresponding diol
and silylation; Dexsil column as Figure 7.
Figure 9 (a) The dense covering of epicuticular wax platelets that
covers the adaxial surface of pea leaves (4600); it is comprised mainly
of C26 and C28 n-primary alcohols. (b) GC analysis as in Figure 7. It
should be noted that there is a completely different wax morphology
and chemistry on the reverse side of the same leaf, viz., crenulate
ribbons and filaments composed mainly of C31 n-alkane.

Chemical Changes during Development
The amounts and composition of the epicuticular waxes on
leaves and fruits may change substantially and continuously
during the course of a plant’s growth. Wax production is
most rapid in the earlier stages of development, but it grad-
ually declines as the organ reaches maturity. If damaged, wax
layers can be regenerated but this ability is usually confined
to the youngest tissues during their normal developmental
phase of wax production. Detailed studies of the ontogeny
of waxes on the surfaces of several species have been pub-
lished and include the leaves of wheat, corn, peach (Prunus
persica), beech (Fagus sylvatica), and lime (Tilia tomentosa),
as well as tomato (Lycopersicon esculentum) and Citrus fruits.
 Secondary Products j Epicuticular Waxes 381

(a) (b)

Nonacosane

FID response
15-Nonacosanone

15-Nonacosanol
Hentriacontane

370°C 310°C 250°C 190°C 130°C

40 30 20 10 0
RT (min)

(c) (d)

Nonacosane

FID response
15-Nonacosanone

15-Nonacosanol

3-Methylnonacosane

370°C 310°C 250°C 190°C 130°C

40 30 20 10 0
RT (min)

(e) (f)

Triacontanal

15-Nonacosanone
Nonacosane
br-Monoesters
C46 C44
C43

370°C 310°C 250°C 190°C 130°C

40 30 20 10 0
RT (min)

Figure 10 Effects of two mutations on the ultrastructure and composition of the epicuticular waxes on the abaxial leaf surfaces of oilseed rape
(Brassica napus). The surface of Nilla normal plants shows the morphology (a, 4700) and chemistry (b) characteristic of most species in the
cabbage family. Wax ultrastructure on the Nilla mutant (c, 5400) is drastically altered in comparison with the normal type, showing extensive plate-
like formations covering and extending upwards from the cuticle surface. However, the only change that can be detected in wax composition (d) is
the production of a small amount of branched (br) alkane. In the Rigo mutant (e), there are extensive modifications to both the crystal structure as
well as the chemistry (f) of the epicuticular wax. Wax production in this mutant is partially inhibited and switches to the formation of n-aldehydes;
this is manifested by the appearance of sparsely distributed short tubular microcrystals. RT, retention time. GC details for all as described in Figure 7.
382 Secondary Products j Epicuticular Waxes
(a)
(c)
Figure 11 Epicuticular waxes that recrystallize in the same physical form as observed in planta. (a) Wax on the flag leaf sheath of barley in situ
(4500) and (b) the isolated wax in vivo (4300). The tubular wax crystals are composed mainly of b-diketones and hydroxy-b-diketones. (c) Wax
on the adaxial surface of a vegetative leaf of barley in situ (9000) and (d) the isolated wax in vivo (10500). n-Hexacosanol is the principal constit-
uent of the wax platelets.
In most cases, major changes were detected in the relative
proportions of the various wax classes identified, some of
which were absent in the early stages, but that later became
major constituents; the reverse also occurred in some species.
In addition, the ratio between cyclic and aliphatic constitu-
ents, if both classes were present, fluctuated markedly. There
was also a marked tendency for homolog chain lengths of
aliphatic classes to increase with the maturity of the wax
layer. For example, in tomato, n- and br-alkanes were the
main components of immature fruits, but these were
replaced by pentacyclic triterpenols and flavonoids in mature
ones; br-alkanes were not detected in the wax of ripe fruits
and the chain-length distribution of the n-alkanes shifted
more toward C31. In beech, primary alcohols are dominant
on juvenile leaves, alkanes and phenolic esters on interme-
diate stages, and aldehydes on mature leaves. The switch in
the production of primary alcohols to alkyl esters during
the development of corn leaves is also accompanied by
a dramatic change in wax microstructure. It should be noted
that some of the ontogenetic changes in wax composition
that have been observed within one species are equivalent
to the gross differences that are reported between different
species.
(b)
(d)
Effects of Environment and Other External Factors
Various abiotic factors have been shown to have a profound
effect on the production, composition, and microstructure of
the epicuticular waxes of a number of plant species. Thus,
wax production is often stimulated by exposure to high levels
of solar radiation, elevated temperatures, and low humidities;
concurrent changes in wax class compositions and homolog
distributions within individual classes may also be detected,
reflecting, in turn, effects on fundamental biosynthetic
processes. However, these chemical changes may not always
be mirrored by modifications to the crystalline form of the
resultant wax deposits. Environmental effects on wax ultra-
structure are well documented for leaves on normal and
mutant forms of B. oleracea.
Most wax constituents are resistant to chemical modifica-
tion under normal environmental conditions. However, alde-
hydes, which are readily oxidized to the corresponding
alkanoic acids, are exceptions; this transformation can be
detected during the course of a growing season on the leaves
of beech for example. The deterioration of waxes often
observed on conifer needles is a symptom that is usually attrib-
uted to air pollution. Although the secondary alcohol tube
 Secondary Products j Epicuticular Waxes 383

(a) (b)

(c) (d)

Figure 12 Epicuticular waxes that do not recrystallize in the same form as observed in planta. (a) On the abaxial surface of peach leaf, the wax layer
is essentially amorphous (1350); (b) after extraction and recrystallization it forms dense masses of platelets (2900). The main constituents of this
wax are triterpenoid acids and n-alkanes. (c) Characteristic tubular forms of epicuticular wax occur on the adaxial surface of Brussels sprout (Brassica
oleracea var. gemmifera) leaves (5600), in marked contrast to the short stubby rodlets that appear following recrystallization of the isolated wax
(d, 6600). The wax from this species is predominantly a mixture of n-nonacosane, n-15-nonacosanone, and n-15-nonacosanol.

waxes present can be oxidized by nitrogen dioxide (NO2)
in vitro to yield oxidized analogs, such as n-10-nonacosanone
and alkanoic acids, that have planar rather than tubular crystal
morphologies, the NO2 concentrations necessary are well
above those that occur even in a heavily polluted atmosphere.
On the other hand, long-term exposure to atmospheric pollut-
ants, such as acid rain, oxides of nitrogen (Nox), sulfur dioxide
(SO2), and ozone (O3), probably has a direct effect on wax
biosynthesis, especially that in the needles of conifer species,
resulting in major changes in wax morphology and coverage.
A number of herbicides, including trichloroacetic acid (TCA),
ethyl N,N-dipropylthiocarbamate (EPTC), diallate, and ethofu-
mesate, are specific inhibitors of cuticular lipid biosynthesis
and are active in the vapor phase. The visible appearance of
plants, such as peas and brassicas, treated with sublethal doses
of these compounds gradually changes from glaucous to glossy,
as wax production and crystallite formation are suppressed.
Epicuticular waxes, especially those of a finely crystalline
nature, may be damaged or even removed by weathering
from strong winds or heavy rains. However, in the majority
of plant species, repairs to any wax layers that have been
damaged can only take place during the period of organ expan-
sion. Once expansion is completed, synthesis of wax ceases
and the layers are then subjected to a variety of environmental
hazards for the rest of the life of the organ. Thus, the epicutic-
ular wax layers may accumulate detritus, become habitats for
microorganisms, or suffer further abrasion and smoothing by
mutual contact with adjacent organs on the same plant or its
close neighbors. Ballistic impacts of precipitation and wind-
blown particles are also known to disrupt epicuticular ultra-
structure. All of these processes can alter the intrinsic nature
of the wax layer, increasing its wettability and permeability,
as well as reducing its reflectivity and anti-adhesive properties.
Such effects are particularly important in evergreen species with
long-lived leaves. In conifers, for example, needle surfaces may
become so densely colonized by epiphytic fungi and algae
within 2 or 3 years that the original wax layer is replaced almost
entirely by a new surrogate surface with entirely different phys-
icochemical properties.
Role in Water Repellency
Epicuticular wax layers have a considerable influence on the
wettability of plant surfaces in contrast to the corresponding
intracuticular waxes, which control their permeability to water.
384 Secondary Products j Epicuticular Waxes

(a) (b)

(c) (d)

(e) (f)

Figure 13 Isolated wax fractions and pure compounds after recrystallization. (a) Purified n-10-nonacosanol (5000) and (b) synthetic n-15-
nonacosanol (5500); the asymmetrical secondary alcohol (a) produces tubes, as opposed to plates for the corresponding symmetrical compound
(b). (c) The n-secondary alcohol fraction isolated from the epicuticular wax of greater celandine (Chelodonium majus) leaves recrystallizes in the same
tubular form (6500) as the wax in situ and in vivo. However, without the secondary alcohol component, the isolated wax recrystallizes as large
aggregates of crenulate plates (d, 1800). Recrystallized n-primary alcohols (e, 6200) and n-aldehydes (f, 6100) isolated from the leaf wax of
pea, showing platelet and rodlet types of crystal morphology, respectively.

The degree of surface wetness is functionally important to plants
in several ways. For example, highly hydrophobic surfaces
discourage the adhesion of dirt and other particulate contami-
nants falling in rainwater. Because most of these extraneous
materials are more hydrophilic than waxes, they adhere preferen-
tially to water droplets, which can then roll off the plant surface.
On the other hand, the presence of water films on leaves
impedes the transport of carbon dioxide and, ultimately, inhibits
photosynthesis. In addition, excess water on the surface may
infiltrate through open stomatal pores and damage the under-
lying mesophyll cells, providing, in turn, a medium for the
growth of invading bacteria and fungi. Plant surfaces are also
the targets for applications of agrochemicals and hydrophobicity
plays an important part in the efficiency of deposition, retention,
 Secondary Products j Epicuticular Waxes 385

and spreading of impacting spray droplets. Most pesticide

formulations contain added surfactants (surface-active agents) Air
in order to reduce the surface tension of the spray liquid and
thereby improve their performance on difficult-to-wet crops
and weeds. Water has an equilibrium surface tension of Liquid
72 mN m1, but addition of an appropriate organosilicone
surfactant can decrease this to about 20 mN m1, thus, enabling Solid
complete wetting of all plant target surfaces.
It is well known that plant waxes are hydrophobic
substances; this property is attributable to the orientation of their Magnitude of contact angle (θ)
molecules in the solid state. X-ray and electron diffraction
studies of aliphatic constituents reveal that their long chains
are arranged perpendicularly or inclined to certain planes, in
individual monomolecular layers. Long-chain alkanoic acids
and primary alcohols are dimeric in the solid state, their
adjacent polar end groups being sandwiched between the
hydrocarbon chains. The net result of these molecular
orientations is that low free surface energy methyl groups are
exposed at an air–solid interface. Maximum hydrophobicity
occurs with n-alkanes, where the terminal methyl groups are Surface Surface
arranged in the tightest possible packing. The situation is less chemistry roughness
clear with the molecular composition of the surfaces of solid
cyclic wax components but they tend to be more wettable than
their aliphatic counterparts.
The relative wettabilities of isolated epicuticular waxes, as
well as plant surfaces, can be quantified by the contact angles
formed by water, viz., the angle between the solid surface and Air−solid
a tangent drawn to the liquid surface where it meets the solid interfaces
(Figure 14). Such measurements provide an inverse measure
of the adhesion between a solid surface and a liquid, with
Figure 14 Summary of the main physicochemical factors governing
complete wetting ¼ 0 and repellency ¼ 180 . When isolated
the wettability of plant surfaces. The liquid droplet shown exhibits
and reconstituted as a smooth layer, most waxes and single
a contact angle of about 801 on the surface of the solid.
wax components show contact angles only between about 95
and 110 , the latter being recorded for n-alkanes. However, the
surfaces of plants exhibit a much wider range of wettabilities rough. At the 100-mm scale, there are trichomes and papillate
with contact angles being as low as 45 up to 150 and above; epidermal cells, with ridged reticulate patterning on the cell
the important modifying factor present on plants is microrough- surface discernible at the 10-mm level, together with wax crys-
ness. If a surface has a contact angle less than 90 , this suggests tallites 1 mm in size. Many insect wings and bird feathers also
that it has an incomplete covering of wax or possesses other display an array of chemical and macroscopic/microscopic
surface features such as corrugations or trichomes that enhance features that contribute to their highly water-repellent nature
wetting. It is well known that the hydrophobicity of a waxy the proverbial water off a duck’s back.
surface is increased by roughening it, and this provides an expla-
nation for plant surfaces with contact angles greater than 110 .
The most water-repellent plant surfaces known are those that Possible Functions in Pest and Disease Resistance
have well-defined microcrystalline epicuticular wax deposits.
The effective surface areas of these layers are several orders of It is generally agreed that the intrinsic chemical and morpholog-
magnitude greater than that of a corresponding smooth layer ical features of plant surfaces are important signals influencing
and, in turn, cause the entrapment of air films between water the behavior of phytophagous and entomophagous insects, as
droplets and the cuticle surface; the contact angle between air well as the infectivity of fungal pathogens. However, attempts
and water is 180 . The contribution of a wax layer to water repel- to establish correlations between susceptibility or resistance of
lency can be readily demonstrated by the observation of plant species to damage by these biological agents, with either
a substantial decrease in contact angle after its removal from the composition or fine structure of the corresponding epicutic-
the plant surface by gentle abrasion or solvent washing. In ular waxes, have met with limited success. Most of the work has
addition, it is possible to calculate the fraction of air–liquid been done using cultivars or mutant forms of important crop
interface for a difficult-to-wet surface from the contact angle species, particularly B. oleracea and Z. mays; nevertheless,
recorded on the surface and that obtained from a smooth layer much of the published information is either circumstantial or
of the wax isolated from the same surface. anecdotal. Part of the problem is thought to arise from compli-
It should be noted, in passing, that some of the most water- cations caused by the presence of other stimulatory or inhibi-
repellent leaves known in nature, such as those of the floating tory organic compounds, e.g., sugar esters, amino acids, and
ferns Salvinia and Azolla, and the lotus Nelumbo, are extremely volatile diterpenoids, also secreted onto the cuticle surface or
386 Secondary Products j Epicuticular Waxes
even dissolving in the layer of epicuticular wax. This is especially
important for leaves that possess glandular trichomes, such as
tobacco (Nicotiana tabacum) and potato (Solanum tuberosum),
or vesicles, as in the case of lupin (Lupinus albus).
In some plant species, epicuticular waxes may be instru-
mental in providing some of the visual, tactile, or chemical
signals necessary for the initial attachment and subsequent
movement, feeding, and oviposition of herbivorous insects,
as well as other insects that prey on them. For example, micro-
crystalline waxes, particularly if they occur in a flat plate-like
form, may be exfoliated by insects walking over the surface
and thus could deter their attachment due to the lack of adhe-
sion. Another possible physical mechanism for defense or
attraction involves the degree of scattering of incident light
by the waxy blooms of glaucous species; this effectively alters
the visible appearance of the surface to prospective insect pests
and may influence host selection. Some insects, such as locusts,
are known to palpate the foliage of plants prior to feeding, and
bait tests have confirmed that most of the commonly found
aliphatic epicuticular wax constituents stimulate acceptance
behavior, whereas others, such as the pentacyclic triterpenoids,
a and b-amyrin, act as deterrents. The effects on growth and
reproduction of ingesting the epicuticular wax from Z. mays
leaves and classes of constituents isolated from it have been
examined in detail for the fall armyworm (Spodoptera frugi-
perda) using artificial diets. In most cases, a significant increase
in the growth rates of larvae was observed.
There have been few direct attempts to examine the influ-
ence of epicuticular waxes on the developmental sequences
leading from spore adhesion to germination and infection by
plant pathogenic fungi. It is known that surface hydrophobicity
is an important factor for some pathogens, but not for others,
although many spores will germinate quite readily on artificial
surfaces, such as glass and cellophane. However, the success or
failure to develop viable infection structures subsequently may
be dependent on the recognition of specific chemical or
morphological clues. A cogent example of this phenomenon
is Erysiphe graminis on ryegrass (Lolium spp.) leaves, which
develops normally on the adaxial surface but not on the corre-
sponding abaxial surface. The wax on the adaxial surface is rich
in primary alcohols and plate-like in form, whereas that on the
abaxial surface occurs as thin amorphous sheets composed
mainly of aldehydes. This could provide a possible explanation
for the resistance of the latter surface to infection by the
powdery mildew, especially since germlings of this fungus
developed normally when the wax layer was removed.
Economic Importance
Waxes obtained from a number of vegetable sources have had
a long history in commerce worldwide and the reader is
referred to the comprehensive treatise by Warth. They have
found uses in the food, cosmetic, candle, rubber, adhesives,
paper, printing, and electrical industries, but are most highly
valued as essential ingredients in the manufacture of high-
quality polishes for a wide variety of materials. Although
many of the traditional vegetable waxes are no longer
produced commercially, having been replaced by synthetic
or semisynthetic substitutes, there are three that are still of
economic importance. The most valuable is carnauba isolated
from the leaves of a Brazilian palm (Copernicia cerifera); it is
one of the hardest waxes known with a melting point of
85  C and is composed mainly of very long-chain alkyl esters.
Another Brazilian palm wax, ouricuri, is used as a substitute for
carnauba and is obtained from the leaves of Syagrus coronata,
Cocos coronata, or Attalea excelsa. Finally, there is candelilla
from Mexico, the wax isolated from the shrubs Euphorbia anti-
syphilitica and E. cerifera. It differs in composition from the
two types of palm waxes in having a high content of nalkanes,
with C31 as the dominant homolog. Japan wax extracted from
the berries of Toxicodendron spp. is also still on the market, but
it is not a true wax because it is a glycerol-based vegetable
tallow.
See also: Crop Diseases and Pests: Bacterial Diseases; Fungal
and Oomycete Diseases. Physiology: Basic Water Relations.
Plant Cells: Leaf Development. Secondary Products: Wax
Pathways.
Further Reading
Barthlott, W., Neinhuis, C., Cutler, D., et al., 1998. Classification and terminology of
plant epicuticular waxes. Bot. J. Linn. Soc. 126, 237–260.
Cutler, D.F., Alvin, K.L., Price, C.E. (Eds.), 1982. The Plant Cuticle. Academic Press,
London.
Gould, R.F. (Ed.), 1964. Contact Angle, Wettability and Adhesion. American Chemical
Society, Washington, DC.
Hamilton, R.J. (Ed.), 1995. Waxes: Chemistry, Molecular Biology and Functions. Oily
Press, Dundee.
Harwood, J.L., Boyer, J. (Eds.), 1990. Lipids, Membranes and Aspects of Photobi-
ology. Academic Press, London.
Holloway, P.J., 1970. Surface factors affecting the wetting of leaves. Pestic. Sci. 1,
156–163.
Holloway, P.J., Rees, R.T., Stock, D. (Eds.), 1994. Interactions between Adjuvants,
Agrochemicals and Target Organisms. Springer-Verlag, Berlin.
Juniper, B.E., Jeffree, C.E., 1983. Plant Surfaces. Edward Arnold, London.
Juniper, B.E., Southwood, R., 1986. Insects and Plant Surfaces. Edward Arnold,
London.
Kerstiens, G. (Ed.), 1996. Plant Cuticles: An Integrated Functional Approach. BIOS
Scientific Publishers, Oxford.
Kolattukudy, P.E. (Ed.), 1976. Chemistry and Biochemistry of Natural Waxes. Elsevier,
Amsterdam.
CRC handbook of chromatography. In: Mangold, H.K. (Ed.), 1984. Lipids, vol. 1. CRC
Press, Boca Raton.
Martin, J.T., Juniper, B.E., 1970. The Cuticles of Plants. Edward Arnold, London.
Percy, K.E., Cape, J.N., Jagels, R., Simpson, C.J. (Eds.), 1994. Air Pollutants and the
Leaf Cuticle. SpringerVerlag, Berlin.
Warth, A.H., 1956. The Chemistry and Technology of Waxes. Reinhold Publishing,
New York.