4
Plant Waxes
Introduction
Biosynthesis
Taxonomic Usefulness
Function of Waxes
Commercial Value of Waxes
Biological Activity
Chemical Fossils and Petroleum
Cutin and Suberin
Analysis of Waxes
References
INTRODUCTION
Plant waxes are complex mixtures of hydrocarbons, alco-
hols, aldehydes, ketones, esters, acids, and combinations of
these that are deposited in a layer outside the epidermal cells.
This complex mixture of lipids probably is synthesized in
the epidermal cells of most plants and exuded onto the sur-
face. Plants usually possess cutin, a layer of cross-esterified
hydroxy fatty acids which is also deposited on the surface
of the epidermal cells (Kolattukudy, 1980). This layer is
impregnated with waxes. The combined lipid covering not
only protects the plant from invasion by foreign organisms
but also helps to regulate transpiration.
Waxes are usually isolated by boiling, scraping, or wash-
ing the outside surface with a nonpolar solvent. Surface lip-
ids differ markedly from both seed lipids and other leaf lip-
ids. (See Chapter 2.)
BIOSYl'l'l1lESIS
Most components of the wax layer are derived from long-
chain fatty acids (C2.-C34 ) formed by chain extension of
shorter-chain fatty acids (Fig. 4.1). The shorter-chain fatty
D. S. Seigler, Plant Secondary Metabolism
© Springer Science+Business Media New York 1998
acid precursor seem to be formed in the same manner as
those previously discussed. Chain elongation requires malo-
nyl-CoA and NADPH as the elongating agent and reductant,
respectively; exogenous stearic acid can be incorporated.
Stearyl-CoA, but not stearyl-ACP, has been demonstrated
to be elongated by endoplasmic reticulum preparations from
the epidermis of leeks, Allium porrum (LiJiaceae). The
length of acids produced depends on the plant species, but
C2., C30 , and C32 acids are commonly encountered (Kolattu-
kudy, 1980).
Long-chain alcohols are formed by reduction of long-
chain fatty acids. A fatty acyl-CoA reductase has been dem-
onstrated in cabbage, Brassica oleracea (Brassicaceae).
Fractionation of the protein fraction isolated in these studies
indicated that one protein fraction catalyzed acyl-CoA re-
duction to the aldehyde stage with NADH as the preferred
reductant and another catalyzed reduction of the aldehyde
to the alcohol with NADPH as the preferred reductant. The
similarity of the naturally occurring fatty aldehydes and fatty
alcohols of a given plant is probably due to the fact that
aldehydes represent intermediates in the synthesis of fatty
alcohols (Kolattukudy, 1980).
Wax esters in plants are formed by the combination of
long-chain alcohols and long-chain acids. Although this can
occur in several ways, it is most probable that, in vivo, these
esters are formed by action of an acyl-CoA alcohol lransacyl-
ase that has been demonstrated to be present by Kolattukudy
(1980).
Wax esters from jojoba (Simmomisia chinensis, Simmonds-
iaceae) consist of molecules with mostly C20 acid (monoene)
esterified with about an equal mixture of Yo and C22 alco-
hols (monoene) (Yermanos, 1978; 1981; Miwa, 1971). In
studies of the biosynthesis of the fatty acids and alcohols in
slices of fresh jojoba cotyledons, a radioactive label from
glucose was incorporated into all carbons of both the C 20
and C22 acids and alcohols. In contrast, exogenous acetate
was used almost entirely for chain elongation from endoge-
51
S2 Plant Waxes
acelyl-CoA fatty acid syntbetase
+ 7 malonyl-CoA
mnlonyl-CoA
-------I.~
NADPH
slearoyl-ACP
stearoyl.CoA
malonyl-CoA
NADPH
-
NADPH
CH3(CH,),.CH,OH
Fig. 4.1. Biosynthesis of long-chain fatty acids, aldehydes, alcohols. and wax esters. (Kolattukudy. 1980 modified and used with pennission of the
copyright owner. Academic Press. Orlando. FL).
nous oleate (pollard et aI., 1978). In this plant, fonnation of
oleic acid occurs on the ACP track. The acid is released and
subsequent reactions leading to the fonnation of C 2G and Y2
fatty acids and alcohols occur as CoA derivatives.
Hydrocarbons found in the wax layer of plants also are
synthesized from long-chain fatty acids. C ,2 , C ,4 , C ,6 , and
C ,S fatty acids are incorporated into Y9 alkanes. Com-
pounds that are efficient precursors for long-chain fatty acids
are also efficient precursors of hydrocarbons. Both groups
of compounds are synthesized in epidennal cells. Exogenous
C3G fatty acid is converted into C29 alkane in cabbage,
Brassica oleracea (Brassicaceae), and similar results have
now been obtained in a number of other plants. Oxygen
is required for this decarboxylation. Further, an <x-hydroxy
intennediate is involved (Kolattukudy, 1980).
Secondary alcohols, ketones, and ketols also are synthe-
sized from long-chain fatty acids. Ketones generally appear
to be synthesized from secondary alcohols by oxidation. The
mechanistic details of these reactions remain obscure.
In an exceptional case, Pinus jeffreyi essential oils consist
of about 98% n-heptane. Acetate can serve as a precursor
for this compound in the plant.
TAXOI'lOI'lIC VSEFULl'IESS
Virtually all plants have hydrocarbons in the same molecu-
lar-weight range; those of C 29 , C3l , and C33 are essentially
ubiquitous. The same is true for wax esters and other long-
chain lipid constituents of plants. Although a small number
of plants have been examined, there appears to be little varia-
tion that would serve to differentiate plant groups (Eglinton
and Hamilton, 1963).
I'Vl'ICDOI'l OF WAXES
As previously mentioned, waxes function primarily to pro-
tect the plant, by preventing entry of foreign organisms and
desiccation of the plant. However, waxes may play other
palmiloyl-ACP
stearic acid
-
CH,(CHzl,.COCoA
- NADH
' " acyl-CoA: alcohol
~ transacylase
wax eslers
roles. For example, in species of Nepenthes, an insectivorous
plant, wax particles inside the "trap" are easily detached
and stick to the feet of insects, preventing them from getting
a foothold inside the trap and escaping.
COl'lllmRCIAL VALVE OF WAXES
Waxes are of considerable commercial importance, as syn-
thetic materials have not been produced at a price that will
allow them tu compete with the natural substances. The most
important natural waxes are camauba from palms of the
genus Copernicia, candelilla from Euphorbia antisyphlitica,
and sugar cane wax-a by-product of the sugar industry
(Schery, 1972).
Camauba wax is comprised mostly of wax esters, cande-
Iilla contains a significant hydrocarbon fraction, and sugar
cane wax is a complex mixture of materials.
For the last several years, there has been mnch interest
in jojoba, Simmondsia chinensis, a plant native to the south-
western United States and Mexico, because of the presence
of liquid wax esters in the seeds (Wisniak, 1988). Numerons
schemes to convert desert land into orchards of these plants
have been proposed. The oil is an excellent lubricant, has
good emollient properties, and may replace spenn whale oil
for many uses. As the fatty acid combination of Limnanthes
douglasii (meadowfoam, Limnanthaceae) is quite similar to
that of jojoba, the synthesis of a similar wax ester by hydroly-
sis and reduction followed by esterification has been pro-
posed (Miwa and Wolff, 1962).
Hexadecanol and octadecanol have been used extensively
in the sonthwestern United States to produce a monolayer
on the top of lakes and other nonfJowing bodies of water to
retard evaporation. These compounds also have potential
agricultural applications (Taylor et aI., 1964). Gennination
of bluegrass seed was increased by application of these mate-
rials to soil (Atsatt and Bliss, 1963).

BIOLOGICAL ACTIVITY
It is generally assumed that long-chain hydrocarbons are
relatively inert and lack pronounced biological activity.
However, octacosanol (C2S ) has androgenic activity in
chicks (measured by comb growth) similar to 11,000 times
the same amount of testosterone propionate.
The application of similar compounds to crop plants has
been conducted (Taylor et aI., 1964). I-Triacontanol, a
straight-chain fatty alcohol, has been isolated from alfalfa
and is sometimes found in other leaf waxes as the palmitate
ester. This compound has been reported to be a powerful
plant growth stimulant. Application of amounts as low as 4
mg per acre can lead to 10-40% increases in growth and
yield of food crops. Application of this compound to corn
and rice plants produces a response within minutes-even
in the dark. Triacontanol increased hypocotyllength of ger-
minating cucumber seeds by 14% over controls, but it
showed inconsistencies in field tests on agricultural crops.
I-Docosanol reportedly stimulates growth in auxin assays.
Lower homologs with 16-28 c",bon atoms and structural
isomers with other hydroxyl substitutions are ineffective
(Gross, 1980).
Surface waxes of plants are important in contact chemore-
ception by insects (Stadler, 1984, 1986). After being at-
tracted to the plant by visual and olfactory cues, the undam-
aged plant surface is normally the first point of contact. The
physical nature of the surface also is important in determin-
ing whether the insect will accept the plant. Long-chain fatty
acids are involved in the biting behavior of Locusta migra-
toria (Chapman, 1977). Undoubtedly, many more volatile
compounds are dissolved in the surface waxes, but the waxes
themselves also are important for sutface recognition.
Some nest parasites in social insects are protected because
their cuticular chemistry is very similar to that of the hosts.
The complex cuticle hydrocarbon blends of parasite and host
are virtually identical and the hosts cannot distinguish the
parasites from conspecifics (Stowe, 1988). Nest parasites
of the genus Trichopsenius biosynthesize their hydrocarbon
mixture; the pattern matches the species-specific mixture of
their termite hosts. In contrast, the scarabaeid beetle, Myr-
mecaphodius excavatacollis, which associates with ants, ac-
quires its hydrocarbons by association with the host and can
invade the nests of several different species of ants with
different hydrocarbon blends (Stowe, 1988). In another bi-
zarre example, larvae of the neuropteran, Chrysopa slosso-
nae, cover themselves with wax plucked from their wax-
coated aphid prey. The ants that tend these aphids fail to
recognize the neuropterans and do not attack them (Stowe,
1988).
The social wasp. Parachartergus aztecus, an ant Pseu-
domyrmex ferrugineus, and the tree they inhabit, Acacia col-
lins;;, exemplify a complex biological interaction (Espelie
and Hermann, 1988). The ants feed on and protect the tree
from herbivory and infringement by other plants and fungi,
the trees provide a place for the ants and wasps to nest,
Plant Waxes 53
and the ants protect the wasps from predators as well. The
cuticular ratios of alkanes and alkenes of the ants and wasps
were found to be identical. The compounds from these two
insect species proved to be identical to the cuticular hydro-
carbons from the spines of the tree (Espelie and Hermann,
1988).
Alkanes also play an important role in nest mate recogni-
tion of wasps (Polistes metricus). However, these same sig-
nals may announce the wasp's presence to possible predators
(Espelie and Hermann, 1990; Espelie et aI., 1990).
CHEMICAL FOSSILS AND PETROLEUM
A major portion of the petroleum used today is derived from
lipids of plants deposited in past eons. The structures of most
compounds isolated from petroleum suggest a derivation
from fatty acids. Various chemical changes have occurred,
mostly reduction and decarboxylation, so that most petro-
leum is comprised of a mixture of odd- and even-chain-
length hydrocarbons. Some petroleums have a variety of
other compounds that appear to be derived from chemical
modification of other types of secondary metabolites.
These compounds may be viewed as chemical fossils. In
some instances, the mixture of compounds may be correlated
with a particular plant or plant groups, although such correla-
tions are usually difficult in coal and in petroleum.
CUTIN AND SUBERIN
Cutin, the lipid that covers the outside of epidermal cells,
and suberin, which is associated with cork cells in plants,
are largely comprised of cross-linked hydroxy fatty acids.
The composition of each type of compound varies from plant
to plant. Generally, cutin is distinct from suberin in the type
of monomeric units present (Harwood, 1980).
Cutin is composed chiefly of a C I6 andlor a CIS family
of monomers (Fig. 4.2). The usual components of the former
are palmitic acid, 16-hydroxypalmitic acid, and 1O,16-dihy-
droxypalmitic andlor its positional isomers in which the mid-
chain hydroxyl group is at C 9 , C g , or C7 • Monomers that
could be derived from the above acids by further oxidation
or reduction also have been found in cutin from some plants.
The C I8 family consists of stearic acid, oleic acid, linoleic
acid, 18-hydroxyoleic acid, 18-hydroxy-9,IO-epoxystearic
acid, and 9,1O,18-trihydroxystearic acid together with their
11.-12 unsaturated analogs. Members of the C I6 family are
the dominant components of the cutin offast-growing plants,
whereas the cutin of slower-growing plants with a thicker
cuticle consists of a mixture of C16 and CIS monomers (Har-
wood, 1980).
The major aliphatic components of suberin are w-hydroxy
acids, the corresponding dicarboxylic acids, very long acids
(greater than Czo), and similarly long alcohols. Among the
w-hydroxy and dicarboxylic acids, monounsaturated CI8 and
54 Plant Waxes
CH,(CH')'4COZH
I
OH
y = 8, 7, 6, or 5
x+y=13
OH
I
HOzC(CH,).CH(CH,).COzH
OHOH
I I
HO(CH,).CH-CH(CH,),COzH
o
II
HO(CH,).C(CHz).COzH
Fig. 4.2. The major components of cutin (Harwood, 1980; Kolattukudy and Espelie, 1985; modified and used with pennission of the copyright owners,
Academic Press, Orlando and Springer-Verlag, Berlin).
saturated C,. acids are often the dominant components (Har-
wood, 1980).
Both cutin and suberin contain phenolic components that
are presumably esterified to the polymeric matrix. p-Coum-
arlc acid and lesser amounts of ferulic acid are found (Kolat-
tukudy and Espelie, 1985). However, a recent reevaluation
of the composition of suberin indicates that there is no solid
evidence for aliphatic-aromatic linkages, nor is it under-
stood how the phenolic units are joined, nor are their identies
clear (Davin and Lewis, 1992).
Al'IALYSIS OF WAXES
Hydrocarbons from waxes are usually removed by dissolu-
tion in a nonpolar solvent such as purified hexane and ana-
lyzed by capillary gas chromatography. Most common hy-
drocarbons can be identified readily by mass spectrometry
(Bagneres and Morgan, 1990). Analysis of wax esters is
more difficult, as several different compounds in the rather
complex mixtures obtained possess the same molecular
weight. Analysis of these mixtures has been effected by tan-
dem mass spectrometry (MS-MS) methods (Spencer and
Plattner, 1984).
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iHZ(CH,),CH=CH(CH,),COZH
OH
CHz(CH,),GH-CH(CHz),COzH
I
OH
"i
CHz(CH,),CH-CH(CHz),COzH
I I I
OH OHOH
i H
OHC(CHz).CH(CHz),COzH
~HIH ~H~H
HO(CH,).CH-CHCHzCH-CH(CHz),COzH
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