The Plant Journal (2008) 54, 670–683 doi: 10.1111/j.1365-313X.2008.03467.

HARNESSING PLANT BIOMASS FOR BIOFUELS AND BIOMATERIALS

Plant surface lipid biosynthetic pathways and their utility for

metabolic engineering of waxes and hydrocarbon biofuels
Reinhard Jetter1,2,* and Ljerka Kunst1
1
Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada, and
2
Department of Chemistry, University of British Columbia, 6174 University Boulevard, Vancouver, BC V6T 1Z3, Canada

Received 28 November 2007; revised 8 February 2008; accepted 13 February 2008.

*
For correspondence (fax +1 604 822 6089; e-mail jetter@interchange.ubc.ca).

Summary

Due to their unique physical properties, waxes are high-value materials that are used in a variety of industrial
applications. They are generated by chemical synthesis, extracted from fossil sources, or harvested from a
small number of plant and animal species. As a result, the diversity of chemical structures in commercial waxes
is low and so are their yields. These limitations can be overcome by engineering of wax biosynthetic pathways
in the seeds of high-yielding oil crops to produce designer waxes for specific industrial end uses. In this review,
we first summarize the current knowledge regarding the genes and enzymes generating the chemical diversity
of cuticular waxes that accumulate at the surfaces of primary plant organs. We then consider the potential of
cuticle biosynthetic genes for biotechnological wax production, focusing on selected examples of wax ester
chain lengths and isomers. Finally, we discuss the genes/enzymes of cuticular alkane biosynthesis and their
potential in future metabolic engineering of plants for the production of renewable hydrocarbon fuels.

Keywords: cuticular waxes, fatty acid elongation, chain lengths, esters, hydrocarbons, industrial products.

Introduction
Primary plant surfaces are impregnated with waxes pro-
duced by epidermal cells (Riederer and Müller, 2006). These
cuticular waxes are complex mixtures of C20–C34 straight-
chain aliphatics derived from very-long-chain fatty acids
(VLCFAs), and in certain plant species also include alicyclic
and aromatic compounds such as triterpenoids, alkaloids,
phenylpropanoids and flavonoids. Plant cuticular waxes
serve as a protective barrier against water loss, UV light,
pathogens and insects. In addition, they are valuable raw
materials for a variety of industrial applications. Wax mix-
tures derived from different plant sources have unique
chemical compositions that determine their physical
properties, and therefore their potential applications and
industrial value.
At present, cuticular waxes are commercially harvested
from only a small number of plant species, so the structural
diversity of their wax constituents is limited. In addition,
these plant species are mostly grown in tropical areas and
are agronomically not well suited to commercial production.
670
These apparent shortcomings of plant surface wax produc-
tion can be circumvented through genetic engineering
approaches using established high-yielding oil crops as a
platform. By introducing wax biosynthetic pathways into
oilseeds, waxes with optimal chemical compositions for
various specialty markets could be produced, including
high-value lubricants, cosmetics and pharmaceuticals, as
well as high-energy fuels.
In this review, we present the chemical diversity of plant
cuticular wax mixtures and summarize our understanding of
the biosynthetic pathways involved in generating this
diversity; provide an overview of commercial sources and
uses of waxes, and of current limitations of wax production;
discuss how engineering of wax biosynthetic pathways in
target crops might be exploited for the production of novel
waxes with specific chain-length distributions in oilseeds;
and describe how wax biosynthetic pathways can be used in
metabolic engineering of plants for the production of
hydrocarbon biofuels. This information complements recent
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd
 Metabolic engineering of waxes and hydrocarbon biofuels 671
reviews that have focused on the chemical composition
(Jetter et al., 2006), biosynthesis (Kunst et al., 2006) and
biological functions of plant cuticular waxes (Bargel et al.,
2006; Riederer and Müller, 2006).
Plant cuticular wax composition and biosynthesis
Cuticular wax composition varies between different species
and organs essentially in two respects: chain-length distri-
bution and compound class composition (Jetter et al., 2006).
This diversity is established during wax biosynthesis in
epidermal cells (Kunst et al., 2006), and involves two types
of pathways: those for the elongation of fatty acid wax pre-
cursors to assorted chain lengths and those for modifying
them into wax components with various functional groups.
Aliphatic compound classes ubiquitously present in cuticu-
lar wax mixtures are alkanes, primary alcohols, aldehydes
and fatty acids ranging in chain length between 20 and 34
carbons, as well as alkyl esters up to C60 in length (Figure 1).
The cuticular waxes from many plant species comprise
roughly equal amounts of the various compound classes,
Figure 1. Structures of major components occurring in plant cuticular wax
mixtures.
(a) Ubiquitous compound classes lacking functional groups (alkanes) or with
primary functional groups. Typically, series of compounds with wide ranges
of chain lengths are present in these classes. n and m indicate the number of
methylene (CH2) groups, and can range from 18 to 32.
(b) Wax constituents with secondary functional groups accumulate to high
concentrations in the wax of certain plant species, usually with very narrow
chain-length and isomer distributions. Typical chain lengths and isomers are
shown for selected combinations of hydroxyl and carbonyl functionalities.
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Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683
with no particular class predominating. For example,
alkanes, aldehydes, primary alcohols, fatty acids and alkyl
esters each contribute 9–42% of the leaf wax of Zea mays
(Bianchi et al., 1984). In contrast, the wax mixtures from
many other plant species contain high percentages of a
single compound class. Hordeum vulgare leaf wax, for
example, contains 89% of primary alcohols, together with
only 0.2–9% of alkanes, aldehydes, fatty acids and alkyl
esters (Giese, 1975).
Compound chain length
Variation in the chain length of wax compounds is generated
during synthesis of VLCFA wax precursors. This process
involves several enzyme complexes in various cellular
compartments. The first phase, the de novo fatty acid syn-
thesis of C16 and C18 acyl chains, is catalysed by the soluble
fatty acid synthase (FAS) complex localized in the plastid
stroma (Ohlrogge and Browse, 1995; Ohlrogge et al., 1993),
and proceeds through a cycle of four reactions utilizing
intermediates attached to acyl carrier protein (ACP). In each
cycle, comprising the condensation of a C2 moiety origi-
nating from malonyl ACP to acyl ACP, the reduction of
b-ketoacyl ACP, the dehydration of b-hydroxyacyl ACP and
the reduction of trans-D2–enoyl ACP, the acyl chain is
extended by two carbons. Three different FAS complexes
participate in the production of C18 fatty acids in the plastid.
They differ in their b-ketoacyl-acyl carrier protein synthase
(KAS) condensing enzymes, which have strict acyl chain-
length specificities: KASIII (C2–C4; Clough et al., 1992), KASI
(C4–C16) and KASII (C16–C18; Shimakata and Stumpf, 1982).
The two reductases and the dehydratase have no particular
acyl chain-length specificity and are shared by all three
plastidial elongation complexes (Stumpf, 1984).
The second phase (Figure 2), the extension of the C16 and
C18 fatty acids to VLCFA chains, is carried out by fatty acid
elongases (FAE; von Wettstein-Knowles, 1982), multienzyme
complexes bound to the endoplasmic reticulum membrane
(Kunst and Samuels, 2003; Xu et al., 2002; Zheng et al.,
2005). To reach the ER-associated fatty acid elongation sites,
saturated C16 and C18 acyl groups must be hydrolysed from
the ACP by an acyl ACP thioesterase, exported from the
plastid, and esterified to CoA. Two classes of acyl ACP
thioesterases, designated FATA and FATB, have been
described in plants. The FATA class exhibits a strong
preference for 18:1 ACP in vitro, while the FATB thioester-
ases predominantly use saturated fatty acids (Voelker, 1996).
The involvement of the FATB thioesterase in cuticular wax
biosynthesis has been confirmed by analyses of the Arabid-
opsis fatb mutant, which exhibits a major reduction in its
wax load (Bonaventure et al., 2003). The specifics of fatty
acid export from the plastid, CoA esterification and transport
to the ER are not well understood. Fatty acids released from
ACP by a thioesterase in the plastid undergo conversion to
672 Reinhard Jetter and Ljerka Kunst
acyl CoAs by a long-chain acyl CoA synthetase (LACS) in the
outer envelope membrane. Of the nine LACS genes anno-
tated in the Arabidopsis genome (Shockey et al., 2002), only
one, LACS9, has been demonstrated to encode a plastid
envelope enzyme (Schnurr et al., 2002). However, loss of
function of LACS9 does not result in reduced export of acyl
groups from the chloroplast, or a wax-deficient phenotype
(Schnurr et al., 2002), suggesting that the LACS isozyme
primarily responsible for CoA esterification of fatty acids en
route to wax biosynthesis has yet to be identified. Move-
ment of the fatty acyl group from the thioesterase to LACS
has been proposed to occur by some type of facilitated
diffusion (Koo et al., 2004), but the exact mechanism of
transfer is not known. An alternative model for fatty acid
export from the plastid was recently suggested by Bates
et al. (2007). Their radiolabelling studies revealed that 16:0
and 18:1 fatty acids synthesized de novo in the plastid can be
incorporated into phosphatidylcholine (PC), perhaps by
direct acylation of lyso-PC. The acyl groups removed from
PC by acyl editing may then be fed into the acyl CoA
pool. However, mechanistic details and the relevance of
this process for epidermal wax formation have not been
established.
Translocation of fatty acids to the ER, where additional
acyl chain elongation and modification of VLCFAs to diverse
aliphatic wax components take place, appears to involve
plastid-associated membranes (PLAMs; Andersson et al.,
2007). Physical manipulation of GFP-labelled ER strands,
using laser scalpels and optical tweezers, experimentally
verified the intimate connection between the plastid and the
ER of Arabidopsis leaf protoplasts. Therefore, PLAMs have
been proposed to be major routes for lipid transfer between
the two organelles.
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683
Figure 2. Wax biosynthetic pathways.
Repeated cycles of four enzymatic steps first
elongate acyl CoA precursors. They are then
modified by one of (up to) five different reactions
into various compound classes. Preferred chain
lengths are indicated by numbers. Characterized
enzymes catalysing key biosynthetic steps are
shown in blue (CER6, condensing enzyme¼
b-ketoacyl CoA synthase; KCR, b-ketoacyl CoA
reductase; dehydratase, b-hydroxyacyl CoA de-
hydratase; CER10, enoyl CoA reductase; CER4,
fatty acyl CoA reductase; WSD1, wax ester
synthase; MAH1, mid-chain alkane hydroxylase).
Elongation of C16 and C18 fatty acids to VLCFAs
involves cycles of four consecutive enzymatic reactions
analogous to those of the FAS (Figure 2), and results in a
two-carbon extension of the acyl chain per cycle. The
chain lengths of aliphatic wax components are typically in
the range of 20–34 carbons, thus multiple elongation
cycles are needed to extend the acyl chain to its final
length. The differential effects of inhibitors on incorpora-
tion of radiolabelled precursors into wax components of
various chain lengths, and analyses of mutants with
defects in fatty acid elongation, demonstrated that
sequential acyl chain extensions are carried out by several
distinct FAEs with unique substrate chain-length specific-
ities (von Wettstein-Knowles, 1993). Specificity of each
elongation reaction resides in the condensing enzyme of
the FAE complex (Lassner et al., 1996; Millar and Kunst,
1997). Consistent with the requirement for fatty acyl
precursors of diverse chain lengths for the synthesis of
cuticular waxes, a family of 21 FAE condensing enzyme-
like sequences has been identified in the A. thaliana
genome (Dunn et al., 2004). An unrelated ELO-like gene
family of putative condensing enzymes, related to the
Saccharomyces cerevisiae condensing enzymes ELO1,
ELO2 and ELO3, has also been annotated (Dunn et al.,
2004). It is not known how many of these putative
condensing enzymes participate in wax production and
how many different condensing enzymes are needed
for the elongation of a C18 to a C34 fatty acyl CoA, as
single condensing enzymes may catalyse multiple elon-
gation steps. The only wax-specific condensing enzyme
characterized to date is CER6 (Fiebig et al., 2000; Hooker
et al., 2002; Millar et al., 1999), which is involved in the
elongation of fatty acyl CoAs longer than C22.
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 Metabolic engineering of waxes and hydrocarbon biofuels 673

Figure 3. Array of biosynthetic reactions leading to wax esters.

First, the variety of chain lengths is generated by elongation, leading to C22 fatty acid (‘ic’) precursors in seeds (black arrows) and including all chain lengths up to C32
in epidermal cells (orange arrows). Then, individual acyl precursors are reduced to the corresponding alcohols (‘ol’) (green arrows), and alcohols and acyl CoAs of
various chain lengths are combined into esters (blue arrows). Depending on the specificity of the elongase (KCS), acyl reductase (FAR) and ester synthase (WS)
enzymes, various mixtures of ester isomers and chain lengths can be generated. Arabidopsis stem surface wax contains esters with predominantly C16 acyl and C22–
C30 alkyl groups.

Unlike the condensing enzymes, the other three enzyme
activities of the FAE complex, the b-ketoacyl reductase,
b-hydroxyacyl dehydratase and enoyl reductase, are shared
by all VLCFA elongase complexes. Thus, these three
enzymes have broad substrate specificities and generate a
variety of acyl products used to make different classes of
lipids (Millar and Kunst, 1997). Because genetic screens in
Arabidopsis did not result in isolation of mutants defective
in the reductases or the dehydratase, suggesting that these
enzymes are essential and/or functionally redundant (Millar
and Kunst, 1997), genes encoding the b-ketoacyl reductase
and enoyl reductase were cloned by homology to the
corresponding sequences from Saccharomyces cerevisiae
(Beaudoin et al., 2002; Kohlwein et al., 2001). Two b-ketoacyl
reductase (KCR) genes are present in both the A. thaliana
and maize (Zea mays) genomes. The maize genes, named
GL8A and GL8B (Dietrich et al., 2005; Perera et al., 2003; Xu
et al., 2002), are not only expressed in the epidermis, but
also in internal tissues. Attempts to generate double
mutants by crossing gl8a · gl8b failed because embryos
carrying both mutations were not viable. Thus, the KCR has
an essential function in plants, most likely in the production
of sphingolipids (Dietrich et al., 2005).
An A. thaliana single-copy gene was identified as an enoyl
reductase (ECR) candidate. Heterologous expression of the
putative plant ECR gene rescued the temperature-sensitive
lethality of yeast tsc13-1elo2D cells (Gable et al., 2004),
demonstrating that it encodes a functional ECR. The A. tha-
liana ECR gene is ubiquitously expressed, and the protein
physically interacts with the Elo2p and Elo3p condensing
enzymes when expressed in yeast (Gable et al., 2004). The
A. thaliana ECR was shown to be identical to CER10 (Zheng
et al., 2005), the protein defective in one of the original
A. thaliana eceriferum mutants isolated by Koornneef et al.
(1989). These eceriferum (literally ‘not bearing wax’)
mutants lack epicuticular wax crystals and therefore have
glossy green inflorescence stems that can easily be recog-
nized in visual screens. Biochemical analysis of the cer10
mutant demonstrated that the ECR gene product is involved
in the VLCFA elongation that is required for synthesis of all
the VLCFA-containing lipids, including cuticular waxes, seed
triacylglycerides and sphingolipids (Zheng et al., 2005).
Although the plant dehydratase remains unknown, recent
identification of the yeast b-hydroxyacyl dehydratase PHS1
(Denic and Weissman, 2007) should permit cloning and
characterization of this enzyme from plants.
Compound classes
In addition to variations in the chain-length distributions,
cuticular wax mixtures from diverse plants and plant
organs also contain various constituent compound classes.
These compounds vary in the nature and position of the
(typically oxygen-containing) functional groups, with the

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Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683
674 Reinhard Jetter and Ljerka Kunst
extreme case of hydrocarbons that are devoid of functional
groups (Jetter et al., 2006). Five or more parallel reactions
(or pathways), all competing for the VLCFA CoA precursors,
can be envisioned leading to these ubiquitous wax com-
ponents: (i) acyl reduction, (ii) esterification with an alkyl
alcohol, (iii) hydrolysis, (iv) aldehyde formation and (v)
alkane formation (Figure 2). Knowledge of all the wax
biosynthetic reactions will assist in their exploitation for
biotechnological production of individual compounds
and/or mixtures of compounds with specific combinations
of functional groups.
In virtually all vascular plants, wax compound classes
with predominantly even numbers of carbons are produced
by the so-called acyl reduction pathway (Figure 2; Kunst
et al., 2006). The most important of these compounds are
primary alcohols and alkyl esters. The latter are essentially
dimeric compounds, in which the primary alcohols are
bonded to acyl groups, most commonly C16, C18 or VLCFAs
(>C20; Figure 3). Primary alcohols are thus central metabo-
lites of wax biosynthesis, and their formation from VLCFA
CoA esters has been studied extensively.
Two reduction steps are required to transform acyl
precursors into primary alcohols, and aldehydes must
occur as intermediates of the reaction sequence. It has
been much debated whether both reduction steps are
catalysed by one fatty acyl reductase (FAR), or whether two
separate enzymes are necessary for alcohol formation.
There is currently substantial evidence for the existence of
a one-enzyme system in a number of plant species,
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683
including the green alga Euglena gracilis (Kolattukudy,
1970) as well as the angiosperms jojoba (Simmondsia
chinensis; Pollard et al., 1979), pea (Pisum sativum; Vioque
and Kolattukudy, 1997) and A. thaliana (Rowland et al.,
2006). For example, functional expression of genes speci-
fying alcohol-forming FARs from jojoba (Metz et al., 2000)
and A. thaliana (Rowland et al., 2006) in heterologous
systems demonstrated that alcohol biosynthesis from
VLCFAs in these species is carried out by a single alcohol-
forming FAR. In contrast, biochemical feeding experiments
that allowed isolation of an aldehyde intermediate suggest
that the two-step process of alcohol formation operates in
Brassica oleracea (Kolattukudy, 1971). However, similar
biochemical evidence from other species and molecular
information supporting the two-step process in any system
is currently lacking.
It is generally assumed that primary alcohols serve as
precursors for ester biosynthesis. However, detailed analy-
ses of esterified and free alcohols of various mutants of
A. thaliana only recently demonstrated a clear correlation of
alcohol chain lengths in both types of compounds, indicat-
ing that the free alcohols are indeed incorporated into the
wax esters (Lai et al., 2007). In addition, this study revealed
that the levels of free alcohols are limiting for ester forma-
tion. Thus, a pool of primary alcohols, generated in the
A. thaliana epidermal cells, is available either for export
towards the cuticle or for esterification with an acyl CoA.
Other plant species exhibit large variations in compositions
of cuticular wax esters, characterized in some cases by broad
Figure 4. Diversity of acyl and alkyl composi-
tions of wax esters from three plant species.
Waxes were extracted from leaf surfaces and
analysed by GC-MS (n = 3). The relative acyl
composition for each ester chain length was
determined from the abundances of MS frag-
ments [RCO2H2]+, and used to calculate overall
acyl and alkyl distributions across ester chain
lengths.
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 Metabolic engineering of waxes and hydrocarbon biofuels 675
distributions of acyl and/or alkyl moieties and in other cases
by relatively high preferences for certain isomers (Figure 4).
In higher plants, mammals and bacteria, ester biosynthe-
sis is catalysed by one of three classes of wax synthase (WS)
enzymes: jojoba-type WS, mammalian WS, and WS/DGAT
bifunctional enzymes. Jojoba-type WS uses a wide range of
saturated and unsaturated acyl CoAs ranging from C14 to
C24, with 20:1 as the preferred acyl and 18:1 as the preferred
alcohol substrate (Lardizabal et al., 2000). In A. thaliana,
there are 12 wax synthases with high homology to the jojoba
WS, but none have yet been characterized. Mammalian WS
enzymes do not have homologues in plants, and have
highest activities with C12–C16 acyl CoAs and alcohols
shorter than C20 (Cheng and Russell, 2004a,b). A bifunctional
WS/DGAT enzyme from Acinetobacter calcoaceticus has a
preference for C14 and C16 acyl CoA together with C14–C18
alcohols (Stöveken et al., 2005). Nearly a hundred WS/DGAT
homologues have been identified from over 20 other micro-
organisms so far (Wältermann et al., 2007), and ten
sequences in the A. thaliana genome have also been anno-
tated as WS/DGATs. One of these enzymes, WSD1, has been
characterized and shown to be responsible for the formation
of cuticular wax esters in A. thaliana stems (R.J., L.K., F. Li,
X. Wu and A.L. Samuels, University of British Columbia,
Canada, unpublished results). The enzyme utilizes mostly
saturated C16 acyl CoA precursors, showing that this
upstream precursor of wax production must be co-localized
in the cell with the primary alcohols, which are synthesized
far downstream in the wax biosynthetic pathway.
Two additional compound classes with predominantly
even-numbered chain lengths, aldehydes and free fatty
acids, are also found in the wax mixture of most plant
species, albeit usually at relatively low concentrations.
Currently, our knowledge on their biosynthesis is very
limited. Formation of free fatty acids must involve hydrolysis
of the elongated acyl CoA precursors (Figure 2). However, it
is not clear whether this reaction occurs spontaneously or
whether it is enzyme-catalysed. Aldehyde formation
requires reduction of acyl CoA precursors, and may occur
as an intermediate step during alcohol formation (see
above), during alkane formation (see below), or indepen-
dently of either of these pathways (Figure 2). Only a single
wax aldehyde-forming reductase enzyme has been partially
purified to date, and the gene encoding this enzyme has not
been identified (Vioque and Kolattukudy, 1997).
A separate set of wax biosynthetic reactions is responsible
for the formation of compounds with predominantly odd
numbers of carbons (Figure 2). Examples of such compound
classes include the alkanes, secondary alcohols and ketones
that occur together in many wax mixtures and typically
share similar chain-length distributions (Jetter et al., 2006).
Early biochemical experiments led to a model describing the
biosynthesis of these compounds as a two-stage process,
with a first set of reactions transforming VLCFA precursors
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Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683
into alkanes and a second series of reactions modifying
them into secondary alcohols and ketones (Kolattukudy,
1965). Subsequent experiments confirmed the central role of
alkanes in this pathway (Kolattukudy, 1968; Kolattukudy and
Brown, 1974; Kolattukudy et al., 1974), either as intermedi-
ates en route to mid-chain functionalized compounds or as
end products if the downstream reactions are missing.
Overall, the second stage of the pathway is relatively
well characterized, whereas the first part remains poorly
understood.
Although conversion of VLCFA precursors into alkanes
could proceed directly in one reaction, the net acyl decar-
boxylation is apparently brought about by a sequence of
transformations. This multistep pathway is supported by the
fact that a number of different A. thaliana mutants with
alkane-deficient cuticular wax mixtures have been described
(Hannoufa et al., 1993; Jenks et al., 1995; Rashotte et al.,
2001, 2004). Cloning of several of these mutated genes
(CER1, CER2 and CER3/WAX2) revealed that the proteins
they encode contain motifs similar to known biosynthetic
enzymes (Aarts et al., 1995; Ariizumi et al., 2003; Chen et al.,
2003; Kurata et al., 2003; Negruk et al., 1996; Rowland et al.,
2007; Xia et al., 1996). While this suggests a potential
enzymatic role for these proteins, their exact function
remains unknown. Due to this lack of molecular information,
it is currently not possible to predict the exact number of
reaction steps involved in the conversion of acyl precursors
into alkanes, the nature of these steps or the resulting
intermediates.
Two alternative pathways have been proposed for the
conversion of acyl compounds into alkanes, which vary in
the central reaction in which a C1 unit is cleaved off (Bianchi,
1995; Bognar et al., 1984; Chibnall and Piper, 1934). The
difference lies in the nature of the immediate precursor from
which cleavage occurs and whether the C1 unit is CO or CO2
(decarbonylation versus decarboxylation). Only one model,
which describes alkane formation as the decarbonylation of
an aldehyde intermediate, has been tested experimentally to
some extent (Cheesbrough and Kolattukudy, 1984). How-
ever, conclusive molecular genetic and biochemical evi-
dence for either model is lacking, leaving alkane formation
as the least understood part of wax biosynthesis.
In A. thaliana leaves, alkanes are the major odd-num-
bered product, while a high level of secondary alcohols and
ketones accompanies alkanes in the stem wax, as well as in
wax from B. oleracea leaves (Baker, 1974; Jenks et al., 1995).
In these instances, a second stage of the pathway is
additionally involved, transforming alkanes first into sec-
ondary alcohols and then into ketones (Figure 2). This
reaction sequence is well-supported by chemical evidence
correlating chain-length and isomer compositions of all
three compound classes (Jenks et al., 1995), and by bio-
chemical evidence provided by feeding experiments and
detailed studies of label positions in resulting products
676 Reinhard Jetter and Ljerka Kunst
(Kolattukudy and Liu, 1970; Kolattukudy et al., 1971, 1973).
Recently, a reverse genetic approach led to the discovery of
a cytochrome P450 enzyme that is involved in secondary
alcohol and ketone formation in A. thaliana (Greer et al.,
2007). The protein is a mid-chain alkane hydroxylase (MAH1)
catalysing two consecutive reactions by first hydroxylating
the central CH2 group of alkanes, and then probably
re-binding the resulting secondary alcohol for a second
hydroxylation of the same carbon. Overall, this confirms the
original hypothesis that the pathway involves alkanes as
central intermediates that may be further oxidized depend-
ing on plant species and organ.
Waxes from certain taxa and/or organs can also contain
other compound classes (Figure 1), most prominently aro-
matic esters and compounds with two hydroxyl or carbonyl
functions (diols, ketols, ketoaldehydes and diketones; Jetter
et al., 2006). These wax constituents can be regarded as
downstream or side products of the ubiquitous biosynthetic
reactions forming the common product classes as described
above. This implies that additional enzymes, expressed at
high levels in certain plant species, can intercept intermedi-
ates and/or final products of the ubiquitous pathways before
they are exported to the cuticle. As these enzymes can
apparently handle the pre-formed wax compounds, they
could be added in a modular fashion to the standard
pathways in heterologous expression systems. This would
allow stepwise addition and modification of secondary
functional group(s), and substantially increase the chemical
diversity of biotechnologically produced wax mixtures. The
necessary biochemical and molecular genetic information
on the biosynthesis of these compound classes is currently
not available. However, cloning and characterization of the
genes involved may become possible in the near future,
once the standard wax biosynthetic pathways are better
understood in A. thaliana, so that the rapidly growing
genomic information from other species (Pennisi, 2007)
can be further exploited.
With the isolation and characterization of a number of key
genes involved in modification of VLCFA precursors into the
diverse wax compound classes, important information on
the intracellular localization of wax biosynthetic pathways
has emerged. The site of primary alcohol formation appears
to be the ER, as shown by localization of the alcohol-forming
Arabidopsis enzyme CER4 after expression in yeast (Row-
land et al., 2006). This is in contrast with mammalian FARs,
which are associated with peroxisomes (Burdett et al., 1991;
Cheng and Russell, 2004a,b), and therefore the localization
of the CER4 FAR will have to be verified in planta.
Meanwhile, the subsequent enzyme in the wax biosynthetic
pathway, the wax ester synthase WSD1, has been localized
to the ER, (R.J., L.K., F. Li, X. Wu and A.L. Samuels,
University of British Columbia, Canada, unpublished
results). Similarly, the mid-chain alkane hydroxylase MAH1
(CYP96A15), which catalyses the last two steps of the alkane-
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683
pathway in A. thaliana stems, is also confined to the
ER (Greer et al., 2007). These two downstream pathway
enzymes are thus co-localized with the VLCFA-generating
FAEs (Kunst and Samuels, 2003; Xu et al., 2002; Zheng et al.,
2005), and it is very likely that the entire wax biosynthesis
process occurs in a single subcellular compartment. All the
wax biosynthetic enzymes and precursors are therefore
expected to be present in the ER of epidermal cells, leading
to accumulation of all intermediates and products in the
extensive membrane system of this organelle.
Current applications and commercial sources of waxes
Wax applications
From the applied perspective, waxes are defined as mixtures
of lipophilic compounds that are solid at room temperature,
range from transparent to opaque, and are ductile and easy
to polish (Illmann et al., 1983; Warth, 1956). Physical
parameters used to characterize waxes include hardness,
cure speed, melting point or range, pour point, viscosity,
(low) surface tension, adhesive strength, optical transpar-
ency and durability, and thermal expansion coefficient
(Anwar et al., 1999; Imai et al., 2001; Kim and Mahlberg,
1995; Kobayashi et al., 2005; McMillan and Darvell, 2000).
Due to their special properties, waxes are used as
lubricants, adhesives, coatings, sealants, impregnation
materials and adjuvants in formulations of (bio)active com-
pounds. A wide range of commercially important final
products rely on waxes, including automobiles, textiles,
papers and specialty inks, pesticides, candles, plastics
and wood–plastic composites, furniture and shoe polish,
household cleaners, cosmetics, dental treatment products,
drugs (lozenge coating) and food (chewing gum, cheese
packaging, confectionery coating).
Wax sources
To meet the demand for material applications, waxes are
currently generated by chemical syntheses, obtained from
geological deposits originating from past organisms (fossil
waxes) or obtained from living organisms (recent waxes)
(Illmann et al., 1983). The vast majority of these waxes are
based either on alkane or ester structures: synthetic waxes
are mainly generated by the Fischer–Tropsch process
(CO + H2) and olefin (ethylene, propylene) polymerization,
giving rise to mixtures of normal and branched alkanes (Ill-
mann et al., 1983; Schulz, 1999; Warth, 1956). Fossil waxes,
on the other hand, are extracted from crude oil and coal
deposits, yielding alkanes and alkyl ester mixtures (together
with the corresponding free acids and alcohols), respectively
(Illmann et al., 1983; Warth, 1956).
Beeswax and wool wax are the prime commodities of
recent waxes from animal sources (Tulloch, 1971; Warth,
ª 2008 The Authors
 Metabolic engineering of waxes and hydrocarbon biofuels 677
1956), whereas the most important plant sources for com-
mercial production of waxes are carnauba (Copernicia
cerifera), candelilla (Euphorbia cerifera and E. antisyphiliti-
ca), ouricouri (Syagros coronata), sugar cane (Saccharum
sp.) and jojoba (Simmondsia chinensis). All these recent
waxes are relatively rich in aliphatic esters, with varying
overall chain lengths of both acyl and alkyl groups, and
contain characteristic admixtures of cinnamates, hydroxy-
esters and lactones, steryl esters, estolides and alkanes
(Basson and Reynhardt, 1988; Holloway, 1984; Illmann et al.,
1983; Lamberton and Redcliffe, 1960; Regert et al., 2005;
Vandenburg and Wilder, 1967; Warth, 1956).
Chemical diversity of commercial waxes
The chemical diversity that is currently available is relatively
broad for the wax alkanes, which include a wide variety of
isomeric branching patterns and chain lengths. For example,
alkane isomers with various patterns of methyl branches can
be synthesized through polymerization of propylene and/or
co-polymerization of propylene and ethylene (Illmann et al.,
1983; Warth, 1956). Furthermore, broad mixtures of (syn-
thetic and fossil) alkanes with diverse chain lengths can be
distilled into mixtures with desired chain-length ranges and
average carbon numbers, or even purified into single chain
lengths (Illmann et al., 1983; Warth, 1956).
The chemical nature of wax esters allows a similar
diversity of isomers and chain lengths through variation of
acyl and/or alcohol carbon numbers (Figure 3). However,
this diversity is presently not commercially exploited,
because the natural wax sources are characterized by ester
mixtures with relatively narrow ranges of chain lengths in
their acyl and alcohol moieties, and therefore also of overall
ester chain lengths (Illmann et al., 1983). To increase the wax
ester diversity and expand the array of ester applications,
chemical variations beyond those available in the current
sources will have to be explored. Examples of highly
desirable modifications in wax ester structures include
in-chain and x-terminal functional groups on the alkyl
and/or on the acyl chains, as well as further variations in
average chain lengths and chain-length distributions. The
target wax esters will also have to accumulate to high levels
in waxes of the source plants to make them viable industrial
raw materials. These goals can only be accomplished by
genetic engineering of wax biosynthetic pathways in oilseed
crops as described below.
Because of their special chain-length composition, some
of the wax ester mixtures from natural sources have proven
to be important commercial commodities. For example, wax
esters consisting of 20:1 fatty acid bonded to 20:1 and 22:1
alcohols are known to have outstanding lubrication proper-
ties combined with high resistance to hydrolysis and
oxidation (Carlsson, 2006). It has been estimated that there
is a market for millions of tonnes of these esters in
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683
lubrication alone (Carlsson, 2006). However, these special
wax commodities can currently be commercially extracted
only from jojoba seeds, i.e. their production relies on the
agriculture of a single plant species (Carlsson, 2006; Purcell
et al., 2000; Yermanos, 1975). Jojoba cultivation is limited by
special growth conditions and low yields with respect to
time and agricultural area, resulting in the high cost of jojoba
oil and its almost exclusive use for high-value products such
as cosmetics and specialty lubricants. Genetic engineering
of the jojoba-type wax biosynthetic pathway in a conven-
tional oilseed crop would result in a new cost-effective
supply of these wax esters and enable their extensive use.
Exploitation of plant cuticular waxes
With the exception of the esters produced in jojoba seeds, all
other commercial plant waxes are harvested more or less
directly from plant surfaces, where they are deposited by the
epidermal cells. Cuticular wax biosynthesis is largely con-
trolled by developmental genetic programs, resulting in
fairly constant, specific compositions for each plant species
and organ. Plant cuticular waxes are therefore chemically
much more diverse than all the other wax sources, and this
greater chemical diversity goes hand in hand with the vari-
ations in wax physical properties that are desirable for
industrial applications. At present, however, the chemical
diversity of plant cuticular waxes is not being exploited
because waxes are commercially harvested from only a
small number of plant species. For example, the carnauba
palm (Copernicia cerifera) is grown exclusively for cuticular
wax production. Its large leaves are covered by an excep-
tionally thick layer of wax reaching a coverage of
300–1000 lg cm)2 of plant surface (Tulloch, 1976). This
greatly facilitates mechanical wax harvest, but yields only
10–100 kg per hectare (Da Silva et al., 1999; Johnson and
Nair, 1985). The vast majority of other plant species have leaf
wax coverages in the range of 1–100 lg cm)2 (Jetter et al.,
2006), but these low wax amounts can be offset by large
surface areas reached in crop fields (Gower et al., 1999). For
example, wheat fields are estimated to contain approxi-
mately 10–200 kg of wax per hectare (Austin et al., 1986;
Bianchi and Corbellini, 1977). However, substantial invest-
ments would be necessary to make harvesting this wax
source commercially viable. Sugar cane (Saccharum sp.) is
the only crop species from which cuticular wax is currently
exploited as a side-commodity, as wax is easily accessible
by extraction of the filter cakes from sugar production.
Approximately 40–240 kg of wax can be produced per
hectare of sugar cane, assuming average crop yields of
50 000 kg ha)1, with filter cakes amounting to 4% of the
mass and waxes to 2–12% of the filter cake (US patent
3931258; Paturau, 1982; Azzam, 2006; FAOSTAT, 2008).
In addition to the modest wax coverages and the lack of
structural diversity in the wax mixtures, wax utilization from
678 Reinhard Jetter and Ljerka Kunst
plant surfaces is also limited by the poor agronomic
properties and special growth conditions of plant species
currently used for wax production. In order to make the
surface wax a lucrative side-commodity in the future, the
wax coverage and composition of temperate plant species
would have to be genetically manipulated in a controlled
way. This is currently not feasible, however, because neither
the regulation of cuticle-forming genes nor the effect of
changed wax composition on the critical cuticle functions
have been investigated in any detail.
Potential for plant wax production in seeds
The apparent shortcomings of wax production on plant
surfaces can be circumvented by genetic engineering
approaches using established high-yielding oil crops as a
platform. By introducing wax biosynthetic pathways into
oilseeds, waxes with optimal chemical compositions for
various specialty markets could be produced, including
high-value lubricants, cosmetics and pharmaceuticals as
well as high-energy fuels. Potential wax yields from oilseed
engineering can be estimated based on the current yields for
major plant oil commodities. For example, Brassica seed oil
yields are in the range of 500–4000 kg ha)1, with typical
values for Canadian canola between 1500 and 1800 kg ha)1
(FAOSTAT, 2008). Even though wax yields from engineered
oilseed crops will probably be lower than the current oil
amounts, this would represent a several-fold increase over
current surface wax yields. That this estimated potential is
realistic can be seen from comparison with jojoba, the only
species known to accumulate wax in its seeds, which yields
75–750 kg of wax per hectare (Botti et al., 1998).
Metabolic engineering for wax ester production
Metabolic engineering of high-yielding oilseed species is a
rapid, cost-effective and rational approach for mitigating
current limitations in the chemical diversity and yield of
surface wax crops. Success will require the following indi-
vidual steps to be accomplished: (i) elucidation of wax bio-
synthetic pathways, (ii) reconstitution of selected pathways,
one at a time, in transgenic systems, (iii) modification of
other lipid biosynthetic pathways by up- or down-regulation
of certain enzymes, to control flux and to generate appro-
priate product mixtures, and (iv) integration of additional
unique modification steps into pathways. The first step in
this process is nearing completion, and the second step is
currently being attempted. The remaining steps can be
tackled in the near future. The following example illustrates
the whole process. (i) Wax ester biosynthesis is relatively
well understood, with genes cloned and characterized from
at least one plant species for each enzymatic step involved
(Lardizabal et al., 2000; Metz et al., 2000; Rowland et al.,
2006). As the early FAE steps of this pathway are shared with
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683
formation of sphingolipids (Dietrich et al., 2005; Zheng et al.,
2005), which are essential membrane components of all
cells, it is likely that sufficient quantities of VLCFA precursors
for wax ester production will be available in all target species
and tissues. (ii) Heterologous overexpression of a fatty acyl
reductase (FAR) together with a wax ester synthase (WS)
should therefore lead to wax ester formation. (iii) Wax ester
formation can be manipulated to enhance flux or generate
novel products. For example, downregulation of triacyl-
glycerol biosynthesis competing for fatty acid percursors
should increase wax ester production. On the other hand,
up-regulation of steroid biosynthesis should increase the
levels of steryl alcohols and result in greater production of
steryl esters and/or mixtures of wax and steryl esters. (iv) To
further increase the wax ester diversity, additional enzymes
may be co-expressed that would lead to the hydroxylation,
desaturation or other modifications of the hydrocarbon
chains of either the acyl or alkyl moieties.
Proof of concept exists that jojoba-type wax esters (C38–
C44) can be produced at high levels by engineering of
oilseeds (Lardizabal et al., 2000). A recent study concluded
that production of wax esters by introduction of a three-
enzyme biosynthetic pathway in the crucifer Crambe abyss-
inica is a viable enterprise for the EU (Carlsson, 2006). This
may lead to high volume production of wax esters at
substantially reduced cost, and to their use for general
automotive lubrication applications, for example, as
transmission and hydraulic fluids.
Wax esters with a vast array of compositions of constit-
uent fatty acids and alcohols are present in various plant
species (Figures 3 and 4). Unfortunately, it is currently not
clear whether the chain-length compositions of esters from
various plant species are governed by the chain-length
specificities of the enzymes involved and/or by substrate
availability. Chemical evidence for A. thaliana stem wax
showed that epidermal ester biosynthesis was limited by
wax alcohol pools, but the study did not address enzyme
specificity (Lai et al., 2007). Biochemical characterization of
various wax ester synthases is currently under way. Once
the substrate specificities of these enzymes are known, it will
be possible to increase the chemical diversity of wax esters
through introduction of the desired enzymes in transgenic
crops. The various types of wax esters will have unique
properties and will serve as substrates for the production of
high-value specialty lubricants, cosmetics and pharmaceu-
ticals. Additional wax ester diversity can be generated by
engineering of artificial enzymatic steps into pathways that
do not normally occur in nature in a single species, to
introduce novel functional groups in either the acid or
alcohol moieties of the esters.
Examples of novel wax products with broad industrial
applications include esters containing acyl and/or alkyl
moieties with C=C double bonds, cyclopropane rings and
methyl branches. Interestingly, a recent study on wax
ª 2008 The Authors
 Metabolic engineering of waxes and hydrocarbon biofuels 679
hydrocarbons in barley spikes showed that all three struc-
tural features are biosynthetically related (von Wettstein-
Knowles, 2007), implying that a small set of enzymes can
convert elongated wax precursors into these compounds. It
was hypothesized that a desaturase introduces a double
bond with high positional specificity, and then a cyclopro-
pane synthase and/or a methyl transferase generate(s)
branched structure(s). Although the exact nature of the
involved enzymes remains to be determined, their potential
biochemical function makes them very important targets
for cloning and future wax engineering studies.
Esters consisting of C20 and C22 alcohols and a hydroxy
fatty acid, for example ricinoleic acid (18:1 ) OH), are
another type of novel wax product. The presence of hydroxy
fatty acids disrupts the packing of hydrocarbon chains,
thereby reducing the melting temperature of the wax esters
and improving their lubrication properties at low tempera-
tures (Carlsson, 2006).
Wax ester fatty acid and alcohol components are also
valuable industrial raw materials, and wax esters containing
ricinoleic acid would be of particular interest due to high
demand for this chemical as an additive to base oils in
lubricant formulations, and as a feedstock for the manufac-
ture of nylon, surfactants, paints, cosmetics and biodegrad-
able polymers for medical applications (Ogunniy, 2006).
Castor oil, in which nearly 90% of acyl residues are ricinoleic
acid, is currently the only commercial source of hydroxy
fatty acids (Atsmon, 1989; Hogge et al., 1991). However,
castor beans are far from an ideal source, as they require
manual harvesting and contain complex allergens together
with the potent toxin ricin. Castor is also not a temperate
climate crop, making it necessary to import castor oil into
many countries from India and China, with irregular supplies
and fluctuating prices. Engineering of an existing oilseed
crop to replace castor bean as a major source of hydroxy
fatty acids is therefore highly desirable. To date, attempts to
produce oils rich in ricinoleic acid have not been successful
due to a general lack of understanding of the mechanisms
involved in channelling this unusual fatty acid from PC,
where it is synthesized, to storage triacylglycerols (Jaworski
and Cahoon, 2003). In contrast to triacylglycerol synthesis,
the enzymology of wax ester production is not as complex,
so engineering of crop plants that efficiently incorporate
ricinoleic acid into wax esters may be a viable alternative.
To further increase the structural diversity of genetically
engineered wax esters, biosynthetic genes from organisms
other than plants should be considered. Wax esters occur in
a wide variety of organisms (Kolattukudy, 1976), including
mammals (Jakobsson et al., 2006; Yen et al., 2005), birds
(Dekker et al., 2000; Haribal et al., 2005; Sweeney et al.,
2004), fungi (Cooper et al., 2000) and bacteria (Ishige et al.,
2002, 2003). Mammalian and bacterial ester formation is
fairly well understood, providing gene candidates for ester
synthases with various substrate/product chain-length pref-
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683
erences (see description of ester biosynthesis above). Cor-
responding genes from birds, fungi and other organisms,
once they are identified and characterized, will help to
further broaden the repertoire of designer wax esters, for
example with methyl branched moieties. Similarly, genes
involved in the formation of cuticular alkanes of insects
(Howard and Blomquist, 2005) should be explored as
candidates for engineering hydrocarbons (see below).
The biosynthetic pathways for the production of novel
commercial wax esters can be engineered in oilseed crops
that are well suited for their synthesis and utilization. At
present, two crop species are being considered as platforms
for ester production, Crambe abyssinica and Brassica cari-
nata (Carlsson, 2006). These crops are not intended for food
production and will grow wherever other Brassica oilseed
crops are cultivated. C. abyssinica has an advantage over
B. carinata in that it is a high-yielding crop, and does not out-
cross with any other agricultural species (Carlsson, 2006).
However, despite its inferior seed yield and some
out-crossing with other Brassica crops, B. carinata may
have preference because it can be easily and efficiently
transformed.
Possible bottlenecks for wax production in oilseeds will
also have to be addressed for each species, including the
low germination rates of transgenic lines and the intracel-
lular autotoxicity of waxes accumulating in seed embryo
cells, whereas they would be exported to the plant surface
when produced in epidermal cells (Bird et al., 2007; Panik-
ashvili et al., 2007; Pighin et al., 2004). In addition, the
physical behaviour of VLCFA derivatives in seeds will have
to be tested in these transgenic crops. It has to be noted that
jojoba, the only currently available model for seed wax
accumulation, has wax esters with relatively short chain
lengths and a substantial amount of unsaturated acyl and
alcohol moieties. The resulting low melting points make
jojoba wax esters liquid at ambient temperatures. In con-
trast, longer-chain fully saturated esters are solid at room
temperature, and it is not clear whether this might affect
their accumulation in transgenic seeds.
Potential for biotechnological alkane production
Alkanes, the other large group of currently used very-long-
chain wax compounds, can be generated by chemical syn-
thesis and extracted from fossil sources with sufficient
chemical diversity and at very low cost (Schulz, 1999; Warth,
1956). It is therefore not commercially attractive to produce
alkane-rich waxes using biotechnological approaches.
Nevertheless, biosynthesis of cuticular alkanes has great
potential for application in commodities other than waxes.
One important future market for these hydrocarbons is in the
fuel sector, where gasoline and diesel are currently provided
by crude fossil oil consisting of various hydrocarbons. Much
of our transportation system relies on these hydrocarbons,
680 Reinhard Jetter and Ljerka Kunst
because the highly reduced carbon contained in them has
maximum chemical energy. Hydrocarbons can be replaced
to some extent by other compounds including ethanol and
biodiesel (Doran-Peterson et al., 2008; Dyer et al., 2008;
Pauly and Keegstra, 2008), but for some applications (e.g.
aircraft), high-energy hydrocarbon fuels will remain essen-
tial. Therefore, it is highly desirable to develop renewable
hydrocarbon sources.
Production of hydrocarbon-rich biofuels can be accom-
plished by biotechnological approaches harnessing photo-
synthetic organisms. To that end, a number of enzymes have
to be combined so that plant compounds, most importantly
fatty acids, can be utilized and transformed into the desired
products. While the enzymes necessary for the hydrocarbon
assembly have not been characterized from any organism to
date, they are known to be present in the epidermis of higher
plants, where they are capable of converting fatty acids into
the hydrocarbons that accumulate in the cuticular wax
deposited on the plant surface (see above). The cuticular
hydrocarbons are synthesized at too low a level for direct
industrial use. However, this system serves as an ideal
model for studying hydrocarbon formation, and can
be exploited for identification and isolation of genes
and enzymes for bio-gasoline production by genetic
engineering.
The central reaction of the alkane-forming pathway, i.e.
the transition step leading from even- to odd-numbered
carbon chains, is thought to involve the loss (rather than
addition) of one carbon atom from the acyl precursors (see
above). However, the biochemical details of this reaction
have not been determined. Several genes involved in alkane
formation have been cloned (CER1, CER2 and CER3/WAX2),
but all attempts to characterize their protein products have
failed so far. As multiple steps are probably required for
the conversion of fatty acids to hydrocarbons, additional
enzymes and corresponding genes might have to be iden-
tified to complete the pathway. Once all the genes/enzymes
have been established, the knowledge of this pathway can
be exploited for elucidation of analogous pathways leading
to the formation of short- and medium-chain, as well as
branched-chain, hydrocarbons.
Conclusions and perspectives
To overcome current limitations in the supply of waxes, to
generate wax commodities with new physical and chemical
properties that do not normally occur in a single species, and
to create entirely new arrays of wax-derived products with
desired chain lengths and functional groups for the chemical
industry, it will be necessary to harness the wax biosynthetic
diversity present in nature and genetically engineer wax
biosynthetic pathways capable of making such specialty
chemicals. These rationally designed wax biosynthetic
pathways will be introduced into the seeds of oil crops
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683
dedicated to industrial use to avoid threat to the existing
food and feed systems, and will result in production of
renewable, high-value waxes that will be able to compete
with petroleum-based products, thus reducing our depen-
dency on fossil oils. In addition, a better understanding of
cuticular alkane biosynthesis might provide renewable
sources for high-energy hydrocarbons and lead to applica-
tions that do not rely on the physical properties of waxes.
Acknowledgements
This work has been supported by the Natural Sciences and Engi-
neering Research Council (Canada), the Canada Research Chairs
Program, and the Canadian Foundation for Innovation.
References
Aarts, M.G.M., Keijzer, C.J., Stiekema, W.J. and Pereira, A. (1995)
Molecular characterization of the CER1 gene of Arabidopsis
involved in epicuticular wax biosynthesis and pollen fertility.
Plant Cell, 7, 2115–2127.
Andersson, M.X., Goksor, M. and Sandelius, A.S. (2007) Optical
manipulation reveals strong attracting forces at membrane con-
tact sites between endoplasmic reticulum and chloroplasts.
J. Biol. Chem. 282, 1170–1174.
Anwar, M., Khan, H.U., Nautiyal, S.P., Agrawal, K.M. and Rawat,
B.S. (1999) Solubilised waxes and their influence on the flow
properties of lube oil base stocks. Pet. Sci. Technol. 17, 491–501.
Ariizumi, T., Hatakeyama, K., Hinata, K., Sato, S., Kato, T., Tabata, S.
and Troiyama, K. (2003) A novel male-sterile mutant of Arabid-
opsis thaliana, faceless pollen–1, produces pollen with a smooth
surface and an acetolysis-sensitive exine. Plant Mol. Biol. 53, 107–
116.
Atsmon, D. (1989) Castor. In Oil Crops of the World (Robbelen, G.,
Downey, K.R. and Ashri, A., eds). New York: McGraw-Hill, pp.
438–447.
Austin, R.B., Morgan, C.L. and Ford, M.A. (1986) Dry matter yields
and photosynthetic rates of diploid and hexaploid Triticum
species. Ann. Bot. 57, 847–857.
Azzam, A.M. (2006) Separation and analysis of wax from Egyptian
sugar cane filter press cake. Fette Seifen Anstrichmittel, 86, 247–
250.
Baker, E.A. (1974) The influence of environment on leaf wax
development in Brassica oleracea var. gemmifera. New Phytol.
73, 955–966.
Bargel, H., Koch, K., Cerman, Z. and Neinhuis, C. (2006) Structure–
function relationships of the plant cuticle and cuticular waxes – a
smart material? Funct. Plant Biol. 33, 893–910.
Basson, I. and Reynhardt, E.C. (1988) An investigation of the struc-
tures and molecular dynamics of natural waxes: II. carnauba wax.
J. Phys. D, 21, 1429–1433.
Bates, P.D., Ohlrogge, J.B. and Pollard, M. (2007) Incorporation of
newly synthesized fatty acids into cytosolic glycerolipids in pea
leaves occurs via acyl editing. J. Biol. Chem. 282, 31206–31216.
Beaudoin, F., Gable, K., Sayanova, O., Dunn, O. and Napier, J.A.
(2002) A Saccharomyces cerevisiae gene required for heterolo-
gous fatty acid elongase activity encodes a microsomal b-keto-
reductase. J. Biol. Chem. 277, 11481–11488.
Bianchi, G. (1995) Plant waxes. In Waxes: Chemistry, Molecular
Biology and Functions (Hamilton, R.J., ed.). Dundee, UK: The Oily
Press, pp. 175–222.
ª 2008 The Authors
 Metabolic engineering of waxes and hydrocarbon biofuels 681
Bianchi, G. and Corbellini, M. (1977) Epicuticular wax of Triticum
aestivum Demar 4. Phytochemistry, 16, 943–945.
Bianchi, G., Avato, P. and Salamini, F. (1984) Surface waxes from
grain leaves and husks of maize (Zea mays L.). Cereal Chem. 61,
45–47.
Bird, D., Beisson, F., Brigham, A., Shin, J., Greer, S., Jetter, R.,
Kunst, L., Wu, X., Yephremov, A. and Samuels, L. (2007) Char-
acterization of Arabidopsis WBC11/ABCG11, an ATP binding
cassette (ABC) transporter that is required for cuticular lipid
secretion. Plant J. 52, 485–498.
Bognar, A.L., Paliyath, G., Rogers, L. and Kolattukudy, P.E. (1984)
Biosynthesis of alkanes by particulate and solubilized enzyme
preparations from pea leaves (Pisum sativum). Arch. Biochem.
Biophys. 235, 8–17.
Bonaventure, G., Salas, J.J., Pollard, M. and Ohlrogge, J.B. (2003)
Disruption of the FATB gene in Arabidopsis demonstrates an
essential role of saturated fatty acids in plant growth. Plant Cell,
15, 1020–1033.
Botti, C., Prat, L., Palzkill, D. and Canaves, L. (1998) Evaluation of
jojoba clones grown under water and salinity stresses in Chile.
Ind. Crops Prod. 9, 39–45.
Burdett, K., Larkins, L.K., Das, A.K. and Hajra, A.K. (1991) Peroxi-
somal localization of acyl-coenzyme A reductase (long chain
alcohol forming) in guinea pig intestine mucosal cells. J. Biol.
Chem. 266, 12201–12206.
Carlsson, A.S.(2006) Production of Wax Esters in Crambe. EpoBIO
Report. Newbury, UK: CLC Press, http://www.epobio.net/pdfs/
0611CrambeWaxEstersReport_c.pdf.
Cheesbrough, T.M. and Kolattukudy, P.E. (1984) Alkane biosynthe-
sis by decarbonylation of aldehydes catalyzed by a particulate
preparation from Pisum sativum. Proc. Natl Acad. Sci. USA, 81,
6613–6617.
Chen, X., Goodwin, S.M., Boroff, V.L., Liu, X. and Jenks, M.A. (2003)
Cloning and characterization of the WAX2 gene of Arabidopsis
involved in cuticle membrane and wax production. Plant Cell, 15,
1170–1185.
Cheng, J.B. and Russell, D.W. (2004a) Mammalian wax biosynthe-
sis. I. Identification of two fatty acyl-coenzyme A reductases with
different substrate specificities and tissue distributions. J. Biol.
Chem. 279, 37789–37897.
Cheng, J.B. and Russell, D.W. (2004b) Mammalian wax biosynthe-
sis. II. Expression cloning of wax synthase cDNAs encoding a
member of the acyltransferase enzyme family. J. Biol. Chem. 279,
37798–37807.
Chibnall, A.C. and Piper, S.H. (1934) The metabolism of plant and
insect waxes. Biochem. J. 28, 2209–2219.
Clough, R., Matthis, A.L., Barnum, S.R. and Jaworski, J.G. (1992)
Purification and characterization of 3–ketoacyl-acyl carrier protein
synthase from spinach: a condensing enzyme utilizing acetyl-CoA
to initiate fatty acid synthesis. J. Biol. Chem. 267, 20992–20998.
Cooper, L.L.D., Oliver, J.E., de Vilbiss, E.D. and Doss, R.P. (2000)
Lipid composition of the extracellular matrix of Botruytis cinerea.
Phytochemistry, 53, 293–298.
Da Silva, J.A., da Cunha, P.B. and Meunier, I.M.J. (1999) Modelagem
da producao cerifera de carnauba Copernicia prunifera (Miller)
H.E. Moore, no municipio de campo maior – Piaui. CERNE, 5, 61–
68, in Portuguese.
Dekker, M.H.A., Piersma, T. and Sinninghe Damste, J.S. (2000)
Molecular analysis of intact preen waxes of Calidris canutus
(Aves: Scolopacidae) by gas chromatography/mass spectrome-
try. Lipids, 35, 533–541.
Denic, V. and Weissman, J.S. (2007) A molecular caliper mechanism
for determining very long-chain fatty acid length. Cell, 130, 663–
677.
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683
Dietrich, C.R., Perera, M.A.D.N., Yandeau-Nelson, M., Meeley, R.B.,
Nikolau, B.J. and Schnable, P.S. (2005) Characterization of two
GL8 paralogs reveals that the 3–ketoacyl reductase component of
fatty acid elongase is essential for maize (Zea mays L.) develop-
ment. Plant J. 42, 844–861.
Doran-Peterson, J., Cook, D.M. and Brandon, S.K.(2008) Microbial
conversion of sugars from plant biomass to lactic acid or ethanol.
Plant J. 54, 582–592.
Dunn, T.M., Lynch, D.V., Michaelson, L.V. and Napier, J.A.(2004) A
post-genomic approach to understanding sphingolipid metabo-
lism in Arabidopsis thaliana. Ann. Bot. 93, 483–497.
Dyer, J., Stymne, S., Green, A. and Carlsson, A.(2008) High value
oils from plants. Plant J. 54, 640–654.
FAOSTAT (2008) ProdSTAT: Crops. Rome: Food and Agriculture
Organization of the United Nations.
Fiebig, A., Mayfield, J.A., Miley, N.L., Chau, S., Fischer, R.L. and
Preuss, D. (2000) Alterations in CER6, a gene identical to CUT1,
differentially affect long-chain lipid content on the surface of
pollen and stems. Plant Cell, 12, 2001–2008.
Gable, K., Garton, S., Napier, J.A. and Dunn, T.M. (2004) Functional
characterization of the Arabidopsis thaliana orthologue of
Tsc13p, the enoyl reductase of the yeast microsomal fatty acid
elongating system. J. Exp. Bot. 55, 543–545.
Giese, B.N. (1975) Effects of light and temperature on the compo-
sition of epicuticular wax of barley leaves. Phytochemistry, 14,
921–929.
Gower, S.T., Kucharik, C.J. and Norman, J.M. (1999) Direct and
indirect estimation of leaf area index, fAPAR, and net primary
production of terrestrial ecosystems. Remote Sens. Environ. 70,
29–51.
Greer, S., Wen, M., Bird, D., Wu, X., Samuels, L., Kunst, L. and
Jetter, R.(2007) The cytochrome P450 enzyme CYP96A15 is the
mid-chain alkane hydroxylase responsible for formation of sec-
ondary alcohols and ketones in stem cuticular wax of Arabidopsis
thaliana. Plant Physiol. 145, 653–667.
Hannoufa, A., McNevin, J. and Lemieux, B. (1993) Epicuticular
waxes of eceriferum mutants of Arabidopsis thaliana. Phyto-
chemistry, 33, 851–855.
Haribal, M., Dhondt, A.A., Rosane, D. and Rodriguez, E. (2005)
Chemistry of preen gland secretions of passerines: different
pathways to same goal? Why? Chemoecology, 15, 251–260.
Hogge, L.R., Taylor, D.C., Reed, D.W. and Underhill, E.W. (1991)
Characterization of castor bean neutral lipids by mass
spectrometry/mass spectrometry. J. Am. Oil Chem. Soc. 68, 863–
868.
Holloway, P.J.(1984) Surface lipids of plants and animals. In Hand-
book of Chromatography, Lipids. Vol. 1 (Mangold, H.K., Zweig, G.
and Sherma, J. eds). Boca Raton, FL: CRC Press, pp. 347–380.
Hooker, T.S., Millar, A.A. and Kunst, L. (2002) Significance of the
expression of the CER6 condensing enzyme for cuticular wax
production in Arabidopsis. Plant Physiol. 129, 1568–1580.
Howard, R.W. and Blomquist, G.J. (2005) Ecological, behavioral,
and biochemical aspects of insect hydrocarbons. Annu. Rev.
Entomol. 50, 371–393.
Illmann, G., Schmidt, H., Brotz, W., Michalczyk, G., Payer, W.,
Frohning, C.D., Dietsche, W., Hohner, G. and Wildgruber, J. (1983)
Wachse. In Ullmanns Lexikon der Technischen Chemie, Vol. 24.
(Bartholome, E., Biekert, E. and Hellmann, H. eds). Weinheim,
Germany: Wiley VCH, pp. 1–49.
Imai, T., Nakamura, K. and Shibata, M. (2001) Relationships
between the hardness of an oil–wax gel and the surface structure
of the wax crystals. Colloids Surf. A, 194, 233–237.
Ishige, T., Tani, A., Takabe, K., Kawasaki, K., Sakai, Y. and Kato, N.
(2002) Wax ester production from n–alkanes by Acinetobacter sp.
682 Reinhard Jetter and Ljerka Kunst
strain M–1: ultrastructure of cellular inclusions and role of acyl
coenzyme A reductase. Appl. Environ. Microbiol. 68, 1192–1195.
Ishige, T., Tani, A., Sakai, Y. and Kato, N. (2003) Wax ester
production by bacteria. Curr. Opin. Microbiol. 6, 244–250.
Jakobsson, A., Westerberg, R. and Jacobsson, A. (2006) Fatty acid
elongases in mammals: their regulation and roles in metabolism.
Prog. Lipid Res. 45, 237–249.
Jaworski, J. and Cahoon, E.B. (2003) Industrial oils from transgenic
plants. Curr.Opin. Plant Biol. 6, 178–184.
Jenks, M.A., Tuttle, H.A., Eigenbrode, S.D. and Feldmann, K.A.
(1995) Leaf epicuticular waxes of the eceriferum mutants in Ara-
bidopsis. Plant Physiol. 108, 369–377.
Jetter, R., Kunst, L. and Samuels, A.L.(2006) Composition of plant
cuticular waxes. In Biology of the Plant Cuticle (Riederer, M. and
Müller, C.eds). Oxford: Blackwell, pp. 145–181.
Johnson, D.V. and Nair, P.K.R. (1985) Perennial crop-based agro-
forestry systems in Northeast Brazil. Agrofor. Syst. 2, 281–292.
Kim, E.-S. and Mahlberg, P.U. (1995) Glandular cuticle formation in
Cannabis (Cannabaceae). Am. J. Bot. 82, 1207–1214.
Kobayashi, M., Saitoh, M., Ishida, K. and Yachi, H. (2005) Viscosity
properties and molecular structure of lube base oil prepared from
Fischer–Tropsch waxes. J. Jpn Pet. Inst. 48, 365–372.
Kohlwein, S.D., Eder, S., Oh, C.-S., Martin, C.E., Gable, K., Bacikova,
D. and Dunn, T. (2001) Tsc13p is required for fatty acid elongation
and localizes to a novel structure at the nuclear–vacuolar interface
in Saccharomyces cerevisiae. Mol. Cell. Biol. 21, 109–125.
Kolattukudy, P.E. (1965) Biosynthesis of wax in Brassica oleracea.
Biochemistry, 4, 1844–1855.
Kolattukudy, P.E. (1968) Tests whether a head to head condensation
mechanism occurs in the biosynthesis of n–hentriacontane, the
paraffin of spinach and pea leaves. Plant Physiol. 43, 1466–1470.
Kolattukudy, P.E. (1970) Reduction of fatty acids to alcohols by cell-
free preparations of Euglena gracilis. Biochemistry, 9, 1095–1102.
Kolattukudy, P.E. (1971) Enzymatic synthesis of fatty alcohols in
Brassica oleracea. Arch. Biochem. Biophys. 142, 701–709.
Kolattukudy, P.E. (1976) Chemistry and Biochemistry of Natural
Waxes. Amsterdam: Elsevier.
Kolattukudy, P.E. and Brown, L. (1974) Inhibition of cuticular lipid
biosynthesis in Pisum sativum by thiocarbamates. Plant Physiol.
53, 903–906.
Kolattukudy, P.E. and Liu, T.-Y.J. (1970) Direct evidence for bio-
synthetic relationships among hydrocarbons, secondary alcohols
and ketones in Brassica oleracea. Biochem. Biophys. Res. Com-
mun. 41, 1369–1374.
Kolattukudy, P.E., Jaeger, R.H. and Robinson, R. (1971) Biogenesis
of nonacosan-15–one in Brassica oleracea. Phytochemistry, 10,
3047–3051.
Kolattukudy, P.E., Buckner, J.S. and Liu, T.-Y.J. (1973) Biosynthesis
of secondary alcohols and ketones from alkanes. Arch. Biochem.
Biophys. 156, 613–620.
Kolattukudy, P.E., Croteau, R. and Brown, L. (1974) Structure and
biosynthesis of cuticular lipids. Hydroxylation of palmitic acid
and decarboxylation of C28, C30 and C32 acids in Vicia faba
flowers. Plant Physiol. 54, 670–677.
Koo, A.J.K., Ohlrogge, J.B. and Pollard, M.(2004) On the export of
fatty acids from the chloroplast. J. Biol. Chem. 279, 16101–16110.
Koornneef, M., Hanhart, C.J. and Thiel, F. (1989) A genetic and
phenotypic description of eceriferum (cer) mutants in Arabidop-
sis thaliana. J. Hered. 80, 118–122.
Kunst, L. and Samuels, A.L. (2003) Biosynthesis and secretion of
plant cuticular wax. Prog. Lipid Res. 42, 51–80.
Kunst, L., Jetter, R. and Samuels, A.L.(2006) Biosynthesis and
transport of plant cuticular waxes. In Biology of the Plant Cuticle
(Riederer, M. and Müller, C.eds). Oxford: Blackwell, pp. 182–215.
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683
Kurata, T., Kawabata-Awai, C., Sakuradani, E., Shimizu, S., Okada,
K. and Wada, T. (2003) The YORE–YORE gene regulates multiple
aspects of epidermal cell differentiation in Arabidopsis. Plant J.
36, 55–66.
Lai, C., Kunst, L. and Jetter, R. (2007) Composition of alkyl esters in
the cuticular wax on inflorescence stems of Arabidopsis thaliana
cer mutants. Plant J. 50, 189–196.
Lamberton, J.A. and Redcliffe, A.H. (1960) The chemistry of sugar-
cane wax. I. The nature of sugar-cane wax. Aust. J. Chem. 13, 261–
268.
Lardizabal, K.D., Metz, J.G., Sakamoto, T., Hutton, W.C., Pollard,
M.R. and Lassner, M.W. (2000) Purification of a jojoba embryo
wax synthase, cloning of its cDNA, and production of high levels
of wax in seeds of transgenic Arabidopsis. Plant Physiol. 122,
645–655.
Lassner, M.W., Lardizabal, K. and Metz, J.G. (1996) A jojoba
b-ketoacyl-CoA synthase cDNA complements the canola fatty
acid elongation mutation in transgenic plants. Plant Cell, 8, 281–
292.
McMillan, L.C. and Darvell, B.W. (2000) Rheology of dental waxes.
Dent. Mater. 16, 337–350.
Metz, J.G., Pollard, M.R., Anderson, L., Hayes, T.R. and Lassner,
M.W. (2000) Purification of a jojoba embryo fatty acyl-coenzyme A
reductase and expression of its cDNA in high erucic acid rape-
seed. Plant Physiol. 122, 635–644.
Millar, A.A. and Kunst, L. (1997) Very-long-chain fatty acid biosyn-
thesis is controlled through the expression and specificity of the
condensing enzyme. Plant J. 12, 121–131.
Millar, A.A., Clemens, S., Zachgo, S., Giblin, E.M., Taylor, D.C. and
Kunst, L. (1999) CUT1, an Arabidopsis gene required for cuticular
wax biosynthesis and pollen fertility, encodes a very-long-chain
fatty acid condensing enzyme. Plant Cell, 11, 825–838.
Negruk, V., Yang, P., Subramanian, M., McNevin, J.P. and Lemieux,
B. (1996) Molecular cloning and characterization of the CER2 gene
of Arabidopsis thaliana. Plant J. 9, 137–145.
Ogunniy, D.S. (2006) Castor oil: a vital industrial raw material.
Bioresour. Technol. 97, 1086–1091.
Ohlrogge, J. and Browse, J. (1995) Lipid biosynthesis. Plant Cell, 7,
957–970.
Ohlrogge, J.B., Jaworski, J.G. and Post-Beittenmiller, D. (1993)
De novo fatty acid biosynthesis. In Lipid Metabolism in Plants
(Moore, T.S., ed.). Boca Raton, FL: CRC Press, pp. 3–32.
Panikashvili, D., Savaldi-Goldstein, S., Mandel, T., Yifhar, T., Franke,
R.B., Höfer, R., Schreiber, L., Chory, J. and Aharoni, A. (2007) The
Arabidopsis DESPERADO/AtWBC11 transporter is required for
cutin and wax secretion. Plant Physiol. 145, 1345–1360.
Paturau, J.M. (1982) By-Products of the Cane Sugar Industry.
Amsterdam: Elsevier.
Pauly, M. and Keegstra, K.(2008) Cell wall carbohydrates and their
modifications as a resource for biofuels. Plant J. 54, 559–568.
Pennisi, E. (2007) The greening of plant genomics. Science, 317,
317.
Perera, M.A.D.N., Dietrich, C.R., Meeley, R., Schnable, P.S. and
Nikolau, B.J. (2003) Dissecting the maize epicuticular wax
biosynthetic pathway via the characterization of an extensive
collection of glossy mutants. In Advanced Research on Lipids
(Murata, N., Yamada, M., Nishida, I., Okuyama, H., Sekiya, J. and
Hajime, W., eds). Dordrecht, The Netherlands: Kluwer Academic,
pp. 225–228.
Pighin, J.A., Zheng, H., Balakshin, L.J., Goodman, I.P., Western,
T.L., Jetter, R., Kunst, L. and Samuels, A.L. (2004) Plant cuticular
lipid export requires an ABC transporter. Science, 306, 702–704.
Pollard, M.R., McKeon, T., Gupta, L.M. and Stumpf, P.K. (1979)
Studies on biosynthesis of waxes by developing Jojoba seed. 2.
ª 2008 The Authors
 Metabolic engineering of waxes and hydrocarbon biofuels 683
The demonstration of wax biosynthesis by cell-free homogen-
ates. Lipids, 14, 651–662.
Purcell, H.C., Abbott, T.P., Holser, R.A. and Phillips, B.S. (2000)
Simmondsin and wax ester levels in 100 high-yielding jojoba
clones. Ind. Crops Prod. 12, 151–157.
Rashotte, A.M., Jenks, M.A. and Feldmann, K.A. (2001) Cuticular
waxes on eceriferum mutants of Arabidopsis thaliana. Phyto-
chemistry, 57, 115–123.
Rashotte, A.M., Jenks, M.A., Ross, A.S. and Feldmann, K.A. (2004)
Novel eceriferum mutants in Arabidopsis thaliana. Planta, 219, 5–
13.
Regert, M., Langlois, J. and Colinart, S. (2005) Characterisation of
wax works of art by gas chromatographic procedures. J. Chro-
matogr. A, 1091, 124–136.
Riederer, M. and Müller, C. (2006) Biology of the Plant Cuticle.
Oxford: Blackwell.
Rowland, O., Zheng, H., Hepworth, S.R., Lam, P., Jetter, R. and
Kunst, L. (2006) CER4 encodes an alcohol-forming fatty acyl-CoA
reductase involved in cuticular wax production in Arabidopsis.
Plant Physiol. 142, 866–877.
Rowland, O., Lee, R., Franke, R., Schreiber, L. and Kunst, L. (2007)
The CER3 wax biosynthetic gene from Arabidopsis thaliana is
allelic to WAX2/YRE/FLP1. FEBS Lett. 581, 3538–3544.
Schnurr, J.A., Shockey, J.M., de Boer, G.-J. and Browse, J.A. (2002)
Fatty acid export from the chloroplast. Molecular characterization
of a major plastidial acyl-coenzyme A synthetase from Arabid-
opsis. Plant Physiol. 129, 1700–1709.
Schulz, H. (1999) Short history and present trends of Fischer–
Tropsch synthesis. Appl. Catal. A, 186, 3–12.
Shimakata, T. and Stumpf, P.K. (1982) Purification and
characterizations of b-ketoacyl-[acyl-carrier-protein] reductase,
b-hydroxyacyl-[acyl-carrier-protein] dehydrase, and enoyl-[acyl-
carrier-protein] reductase from Spinacia oleracea leaves. Arch.
Biochem. Biophys. 218, 77–91.
Shockey, J.M., Fulda, M.S. and Browse, J.A. (2002) Arabidopsis
contains nine long-chain acyl-coenzyme A synthetase genes that
participate in fatty acid and glycerolipid metabolism. Plant
Physiol. 129, 1710–1722.
Stöveken, T., Kalscheuer, R., Malkus, U., Reichelt, R. and Steinbü-
chel, A. (2005) The wax ester synthase/acyl coenzyme A:diacyl-
glycerol acyltransferase from Acinetobacter sp. strain ADP1:
characterization of a novel type of acyltransferase. J. Bacteriol.
187, 1369–1376.
Stumpf, P.K. (1984) Fatty acid biosynthesis in higher plants. In Fatty
Acid Metabolism and its Regulation (Numa, S., ed.). Amsterdam:
Elsevier, pp. 155–179.
Sweeney, R.J., Lovette, I.J. and Harvey, E.L. (2004) Evolutionary
variation in feather waxes of passerine birds. Auk, 121, 435–
445.
ª 2008 The Authors
Journal compilation ª 2008 Blackwell Publishing Ltd, The Plant Journal, (2008), 54, 670–683
Tulloch, A.P. (1971) Beeswax: structure of the esters and their com-
ponent hydroxy acids and diols. Chem. Phys. Lipids, 6, 235–265.
Tulloch, A.P. (1976) Chemistry of waxes of higher plants. In Chem-
istry and Biochemistry of Natural Waxes (Kolattukudy, P.E., ed.).
Amsterdam: Elsevier, pp. 235–287.
Vandenburg, L.E. and Wilder, E.A. (1967) Aromatic acids of
carnauba wax. J. Am. Oil Chem. Soc. 44, 659–662.
Vioque, J. and Kolattukudy, P.E. (1997) Resolution and purification
of an aldehyde-generating and an alcohol-generating fatty
acyl-CoA reductase from pea leaves (Pisum sativum L.). Arch.
Biochem. Biophys. 340, 64–72.
Voelker, T.A.(1996) Plant acyl-ACP thioesterases: chain-length
determining enzymes in plant fatty acid biosynthesis. In Genetic
Engineering, Vol. 18 (Setlow, J.K.ed.). New York: Plenum Press,
pp. 111–131.
Wältermann, M., Stöveken, T. and Steinbüchel, A. (2007) Key
enzymes for biosynthesis of neutral lipid storage compounds in
prokaryotes: properties, function and occurrence of wax ester
synthases/acyl-CoA:diacylglycerol acyltransferases. Biochemie,
89, 230–242.
Warth, A.H.(1956) The Chemistry and Technology of Waxes. New
York: Reinhold Publishers.
von Wettstein-Knowles, P.(1982) Biosynthesis of epicuticular lipids
as analysed with the aid of gene mutations in barley. In Bio-
chemistry and Metabolism of Plant Lipids (Wintermans, J.F.G.M.
and Kuiper, P.J.C.eds). Amsterdam: Elsevier, pp. 69–78.
von Wettstein-Knowles, P. (1993) Waxes, cutin, and suberin. In
Lipid Metabolism in Plants (Moore, T.S., ed.). Boca Raton, FL:
CRC Press, pp. 128–166.
von Wettstein-Knowles, P. (2007) Analyses of barley spike mutant
waxes identify alkenes, cyclopropanes and internally branched
alkanes with dominating isomers at carbon 9. Plant J. 49, 250–264.
Xia, Y., Nikolau, B.J. and Schnable, P.S. (1996) Cloning and char-
acterization of CER2, an Arabidopsis gene that affects cuticular
wax accumulation. Plant Cell, 8, 1291–1304.
Xu, X., Dietrich, C.R., Lessire, R., Nikolau, B.J. and Schnable, P.S.
(2002) The endoplasmic reticulum-associated maize GL8 protein
is a component of the acyl-coenzyme A elongase involved in the
production of cuticular waxes. Plant Physiol. 128, 924–934.
Yen, C.-L.E., Brown, C.H., IV, Monetti, M. and Farese, R.V., Jr (2005)
A human skin multifunctional O–acyltransferase that catalyzes
the synthesis of acylglycerols, waxes, and retinyl esters. J. Lipid
Res. 46, 2388–2397.
Yermanos, D.M. (1975) Composition of jojoba seed during devel-
opment. J. Am. Oil Chem. Soc. 52, 115–117.
Zheng, H., Rowland, O. and Kunst, L. (2005) Disruptions of the
Arabidopsis enoyl-CoA reductase gene reveal an essential role for
very-long-chain fatty acid synthesis in cell expansion during plant
morphogenesis. Plant Cell, 17, 1467–1481.