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Plant cuticles shine: advances in wax biosynthesis and export
Ljerka Kunst and Lacey Samuels
The plant cuticle is an extracellular lipid structure deposited
over the aerial surfaces of land plants, which seals the shoot
and protects it from biotic and abiotic stresses. It is composed
of cutin polymer matrix and waxes, produced and secreted by
epidermal cells. The use of forward and reverse genetic
approaches in Arabidopsis has led to the identification of
enzymes involved in fatty acid elongation and biosynthesis of
wax components, as well as transporters required for lipid
delivery to the cuticle. However, major questions concerning
alkane formation, intracellular and extracellular wax transport,
regulation of wax deposition, and assembly of cuticular
components into a functional cuticle remain to be resolved.
Address
Department of Botany, University of British Columbia, 6270 University
Boulevard, Vancouver, BC, V6T 1Z4 Canada
Corresponding author: Kunst, Ljerka ([email protected])
Current Opinion in Plant Biology 2009, 12:721–727
This review comes from a themed issue on
Cell Biology
Edited by Jir̆ı́ Friml and Karin Schumacher
Available online 26th October 2009
1369-5266/$ – see front matter
# 2009 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2009.09.009
Introduction
The cuticle is a continuous hydrophobic layer that covers
primary aerial surfaces of all land plants. It plays a critical
role in controlling nonstomatal water loss and is involved
in plant protection against pathogens and insect herbivores. The cuticle consists of two major types of lipids:
cutin, the core structural polymer composed of v- and
mid-chain hydroxy and epoxy C16 and C18 fatty acids and
glycerol, and cuticular waxes, a mixture of mostly
aliphatic very long chain fatty acid (VLCFA) derivatives
and variable amounts of triterpenoids and phenylpropanoids [1,2]. Even though the mechanism of assembly of
these components into a functional cuticle is still largely
unknown, molecular genetic approaches in Arabidopsis
thaliana have led to several major breakthroughs over the
past few years that resulted in a better understanding of
the biosynthesis and export of cuticular lipids. They
include the identification of all the components of the
fatty acid elongation complex involved in the biosynthesis of VLCFA wax precursors, identification of new
enzymes required for the modification of VLCFAs to
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diverse aliphatic wax constituents, localization of these
enzymes to the endoplasmic reticulum (ER) and discovery of new transport proteins for the delivery of waxes to
the plant surface. These recent developments are the
focus of the current review.
VLCFA biosynthesis
VLCFA precursors for cuticular wax production are synthesized in the epidermal cells by the multienzyme fatty
acid elongase (FAE) complex found on the ER. Using
plastid-generated C16 or C18 fatty acids and malonylcoenzyme A (CoA) as substrates, FAE performs a reiterative cycle of four reactions catalyzed by a b-ketoacylCoA synthase (KCS), a b-ketoacyl-CoA reductase (KCR),
a b-hydroxyacyl-CoA dehydratase (HCD), and an enoylCoA reductase (ECR), to produce saturated VLCFAs
with 24–36 carbon atoms (Figure 1). The KCS is the
rate-limiting enzyme of fatty acid elongation and also
determines the length of the final acyl-CoA product
[3,4].
There are two types of KCS enzymes: ELOs, which were
discovered in fatty acid elongation defective mutants of
Saccharomyces cerevisiae [5,6], and FAE1-type KCSs,
which were first characterized in an Arabidopsis mutant
deficient in fatty acid elongation of storage lipids [7]. In
Arabidopsis, four ELO-type KCS sequences have been
annotated [8], but their function has not been experimentally established. In addition, Arabidopsis has a large
family of 21 FAE1-type KCS sequences [8,9], consistent
with cellular requirements for VLCFAs of different chain
lengths. An understanding of how the final acyl chain
length is specified by the KCS enzyme is starting to
emerge. Work by Denic and Weissman [4] on the
ELO-type KCSs in yeast revealed that the determining
factor is the depth of the hydrophobic pocket where the
VLCFA sits during elongation. On the basis of this
finding the authors suggest that once the length of the
growing acyl chain exceeds the depth of the pocket,
further elongation is not possible. However, the confirmation of this hypothesis will require ELO protein
structural information. Whether the mechanism proposed for the ELO KCSs also reflects how FAE1-type
KCS enzymes discriminate between fatty acid substrates
of different chain length remains to be determined. The
development of a proteoliposome system for the reconstitution of VLCFA synthesis in vitro [4] will be a
valuable tool in addressing this issue.
Another important unanswered question concerns the
number of KCS enzymes involved in the elongation of
C18–C36 acyl groups required for wax biosynthesis. Each
Current Opinion in Plant Biology 2009, 12:721–727
722 Cell Biology
Figure 1
Fatty acid elongase (FAE) complex synthesizes very long chain fatty acids (C20 and longer) by repeated cycles of two-carbon addition. In one cycle,
the four enzymes of the FAE complex work sequentially to carry out the condensation of two carbons from malonyl-CoA (carbon donor) to the growing
acyl-CoA chain (carbon acceptor), followed by reduction, dehydration, and another reduction reaction, resulting in a fully saturated acyl-CoA.
KCS usually catalyzes a few successive elongation steps
and various KCSs may have partially overlapping acyl
chain specificities. At present only the ECERIFERUM6
(CER6) FAE1-type KCS has been shown to be waxspecific and essential for the formation of VLCFAs longer
than 22 carbons in Arabidopsis stems [10]. Clearly, other
KCSs that participate in the elongation of VLCFA wax
precursors need to be identified.
called PASTICCINO2 have been found in Arabidopsis
[15,16]. In contrast, there are two KCR orthologs in
the Arabidopsis genome. Surprisingly, only one of these
genes, KCR1, encodes a functional b-ketoacyl reductase
involved in VLCFA biosynthesis [17]. Thus, Arabidopsis
differs from maize, in which two closely related KCRs
(GL8A and GL8B) contribute to the activity of the ERbound elongase [18].
Unlike the KCSs that have strict substrate specificities,
the other three enzymes making up the FAE complex,
the KCR, HCD, and ECR were hypothesized to have
broad substrate specificities, and to be capable of generating the complete repertoire of VLCFAs required by
different classes of lipids in various plant tissues [3,11].
This hypothesis could not be experimentally tested until
recently because the genes encoding these FAE components were not known. Identification of the ECR,
KCR, and HCD in yeast [4,12–14], led to rapid progress
in characterization of the plant enzymes involved in
VLCFA formation. A single ECR gene named ECERIFERUM10 (CER10) and a single gene encoding HCD
Partial loss of KCR1 or PAS2 function is associated with a
general reduction of VLCFA content in seed storage
triacylglycerols and complex sphingolipids, as well as
reduced levels of cuticular waxes. A complete loss of
KCR1 or PAS2 activity is embryo lethal indicating that
the capacity to synthesize VLCFAs is crucial at early
stages of embryo development [16,17]. It is also interesting to note that while KCR1 and PAS2 are both
essential genes in Arabidopsis, the CER10 gene is not,
even though it represents the sole ortholog of the yeast
TSC13 ECR [15]. This suggests that a structurally unrelated but functionally equivalent form(s) of the ECR
must be present in Arabidopsis.
Current Opinion in Plant Biology 2009, 12:721–727
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Cuticular wax biosynthesis and export Kunst and Samuels 723
Biosynthesis of aliphatic wax constituents
Despite the great diversity of chemical compositions
among plant cuticles from different species, various
plant organs and across developmental states, the
aliphatic VLCFA derivatives are ubiquitously present
wax components. They are generated from VLCFAs
by two biosynthetic pathways, the alcohol-forming (acyl
reduction) pathway involved in the production of primary
alcohols and wax esters, and the alkane-forming (decar-
bonylation) pathway, which gives rise to aldehydes,
alkanes, secondary alcohols, and ketones (Figure 2). Even
though most of the major biosynthetic steps leading to
aliphatic wax products have been established both genetically and biochemically, the enzymes that catalyze biosynthetic reactions have not been identified until very
recently. Using the Arabidopsis wax-deficient cer4 mutant,
the CER4 gene was cloned and shown to encode a
fatty acyl-CoA reductase, which catalyzes the two-step
Figure 2
Wax biosynthesis and export of wax component to the plant cuticle. Long chain fatty acyl groups (C16–C18) are generated by a fatty acid synthase
(FAS) in the plastid. They are released by an acyl-ACP thioesterase (FATB), activated to a CoA thioester by a long chain acyl-CoA synthetase (LACS)
and exported from the plastid to the ER. In the ER, fatty acids are extended to very long chain fatty acids (VLCFAs) by a fatty acid elongase (FAE)
comprised of four enzymes as depicted in Figure 1, a b-ketoacyl-CoA synthase (CER6), a b-ketoacyl-CoA reductase (KCR1), a b-hydroxyacyl-CoA
dehydratase (PAS2) and an enoyl-CoA reductase (CER10), and modified to diverse aliphatic wax components. Enzymes of the primary alcohol
pathway, fatty acyl-CoA reductase (CER4) and wax synthase (WSD1) generate primary alcohols and wax esters, respectively. Enzymes and steps
involved in the conversion of VLCFAs to alkanes have not been identified (indicated by question marks). Formation of secondary alcohols and ketones
is catalyzed by a midchain alkane hydroxylase (MAH1). Wax components are mobilized from the ER to the plasma membrane (PM) by an unknown
mechanism and exported from the cell by a process that requires ABC transporters. A recently discovered glysosylphosphatidylinositol-anchored lipid
transfer protein (LTPG) is also involved in wax export to the cuticle, but its precise function has not been determined.
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Current Opinion in Plant Biology 2009, 12:721–727
724 Cell Biology
reduction of VLCFAs to primary alcohols in Arabidopsis
shoots [19]. Primary alcohols together with acyl groups are
then used as substrates for the biosynthesis of alkyl esters
[20], a reaction catalyzed by a wax synthase. A member of
the WAX SYNTHASE/DIACYLGLYCEROL ACYLTRANSFERASE (WS/DGAT) family, homologous to
the WS/DGAT bifunctional protein from Acinetobacter
calcoaceticus, was shown to carry out wax ester formation
in the Arabidopsis stem [21].
In contrast to the enzymes of the alcohol-forming pathway, which have now been identified, the enzymes
involved in the formation of alkanes and the biosynthetic
steps that they catalyze remain enigmatic (Figure 2). A
number of different Arabidopsis mutants with reduced
alkane levels in their stem wax have been described, for
example, cer1, cer2, cer3, cer8, cer19, and cer22 [22,23], but
the genes identified in these mutants have either not
been cloned (CER19, CER22), or if cloned, have not
provided clues about the biochemical role of the proteins
that they encode (CER1, CER2, CER3). An intriguing
new report identifies CER8 as LACS1, a member of the
long chain acyl-CoA synthetase (LACS) family that joins
free fatty acids to CoA [23]. The cer8/lacs1 mutation
results in reduced accumulation of products of the alkane
pathway and a hyperaccumulation of C26–C30 free fatty
acids in the cuticle. Enzyme activity assays using recombinant LACS1 expressed in E. coli demonstrated a corresponding substrate preference for C30 fatty acids.
However, why the CoA activation of VLCFA would be
required for alkane production remains a mystery. The
only currently known enzyme of the alkane-forming
pathway is the recently discovered MAH1, a cytochrome
P450-dependent midchain hydroxylase, which catalyzes
two successive reaction steps, a conversion of alkanes to
secondary alcohols and secondary alcohols to ketones
[24].
Enzymes catalyzing terminal steps of both wax biosynthetic pathways, WSD1 and MAH1, have been localized
in the ER of the epidermis [21,24]. Since all the
VLCFA-forming and all the known wax biosynthetic
enzymes reside in the ER, it is likely that this is the
subcellular compartment where all of the cuticular wax
components are generated.
Cuticular wax export
In epidermal cells, cuticular lipids must be transported
from sites of synthesis on the ER to the plasma membrane (PM), moved through the PM and, finally, translocated across the outer periclinal cell wall to the cuticle
where they apparently self-assemble into the cuticle
proper.
The intracellular traffic of the VLCFAs and their
derivatives is still a matter of speculation. For example,
do the cuticular lipids move in vesicles from the ER to
Current Opinion in Plant Biology 2009, 12:721–727
the Golgi and on to the PM, or is there direct nonvesicular lipid traffic between the ER and PM? Studies
in S. cerevisiae demonstrated the nonvesicular movement of lipids between the PM and the ER [25,26] and
this could occur at membrane contact sites where the
membranes are adjacent but not mixing or fusing
(reviewed by [27]).
The subsequent transport step from the PM to the cell
exterior appears to be carried out by PM bound transporters (Figure 2), although experimental evidence that
the cuticular lipids are the actual transport substrates is
still lacking. Forward genetic studies, using the cer5
mutant [28], identified a PM-localized ATP binding
cassette (ABC) transporter, CER5, required for wax
export from the stem epidermal cells of Arabidopsis
[29]. This ABC transporter of the ABCG subfamily is
related to human ABCG transporters that export a variety
of lipid substrates (reviewed by [30]) and Drosophila eye
pigment transporters such as WHITE, BROWN, and
SCARLET complex (WBC) ABC transporters [31].
The plant ABC transporter nomenclature has recently
been revised and, due to the conserved nature of these
transporters, the Human Genome Organization subfamily
designations could be adopted [32]. For example,
because the Arabidopsis CER5 ABC transporter is a
member of the ABCG subfamily, its name was changed
to ABCG12.
Functional ABC transporters are made up of two ABC
domains and two transmembrane domains (TMD),
which may be present on one polypeptide or on two
‘half-transporter’ polypeptides that contain one ABC
and one TMD domain each. To function, half-transporters can form homodimers, as is the case for the
well-characterized human xenobiotic exporter ABCG2
[30], or heterodimers, such as the Drosophila eye pigment transporters WHITE/BROWN and WHITE/
SCARLET [33]. ABCG12/CER5 could form a homodimer or a heterodimer with one of the other 27 ABCG
half-transporters encoded in the Arabidopsis genome.
Two approaches have been used to look for other
ABCG transporters that could act in cuticular lipid
export: ABC transporters highly expressed in the epidermis during cuticular lipid secretion were characterized using reverse genetics approaches [34,35], or the
expression of all Arabidopsis ABCG half-transporters
was systematically disrupted using RNA interference
(RNAi) [36]. In each case, abcg11 mutants were identified with reduced wax loads on the stem surface,
similar to the abcg12/cer5 mutant lines. In addition,
and in contrast to the abcg12/cer5 mutants, abcg11
mutants displayed organ fusions, a phenotype correlated with disorganized cutin components [37], and
the most severe abcg11 alleles were shown to have
reduced cutin levels in their cuticles [34,35,36]. Surprisingly, the abcg11 mutants were also dwarf and
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Cuticular wax biosynthesis and export Kunst and Samuels 725
sterile, suggesting that the ABCG11 transporter has
more extensive roles than just cuticular lipid export.
sites for defining the steps involved in this regulatory
process.
Double abcg11abcg12 mutants had similar wax defects as
the abcg11 single mutant lines [34], indicating that the
phenotypes were not additive, suggesting that these gene
products may act in the same process or pathway. On the
basis of this genetic evidence, and by analogy with the
Drosophila eye pigment ABC transporters, a working
model has been proposed where heterodimers of
ABCG11 and ABCG12 act in wax transport. In addition,
ABCG11 could dimerize with other partners to translocate diverse substrates, such as cutin precursors [38].
Conflict of interest statement
Once highly hydrophobic wax molecules are extruded
from the PM of epidermal cells, they must somehow cross
the hydrophilic cell wall. It has long been postulated that
cell wall specific carriers, such as lipid transfer proteins
(LTPs), may be involved in the delivery of wax components to the cuticle as LTPs are highly expressed in
Arabidopsis during cuticle formation [39]. Only recently,
the isolation and characterization of an Arabidopsis mutant
disrupted in a glycosylphosphatidylinositol-anchored
LTP, named LTPG, revealed reduced alkane accumulation at the plant surface [40,41], confirming that this
PM-bound LTP is required for lipid export. While LTPG
has lipid binding capacity in vitro [40], the question
remains as to whether this LTP shuttles across the cell
wall to deliver lipids to the cuticle.
References and recommended reading
Conclusions
Cuticular wax deposition on the aerial plant surfaces
requires the coordinated regulation of a complex metabolic network of enzymes and transporters. Molecular
genetic approaches in Arabidopsis have been successfully
employed in perturbing this network at various points to
determine the role of individual proteins in wax biosynthesis, establish the ER as the subcellular compartment
where wax components are generated and identify
proteins involved in wax translocation across the PM to
the apoplast. Gaps remain in our understanding of alkane
formation, the delivery of wax molecules from the ER to
the PM, the mechanism of ABCG-mediated wax export
and the identity of the proteins involved in shuttling wax
between the PM and the cuticle. Finally, the regulation of
cuticular wax deposition in response to developmental
and environmental cues remains obscure. There is good
evidence that wax biosynthesis is under transcriptional
control [22], yet the transcription factors regulating this
process in the epidermis have not been identified. Currently the only known wax regulatory component is the
Arabidopsis CER7 ribonuclease [42], a core subunit of
the exosome, which controls stem wax production via the
alkane-forming pathway. Identification of the mRNA
target of the CER7 ribonuclease, and information on
additional components involved, are essential prerequiwww.sciencedirect.com
The authors are not aware of any biases or conflicts of
interest that might be perceived as affecting the objectivity of this review.
Acknowledgements
The authors acknowledge the artwork of Vicki Earle, UBC Media Group
(Figures 1 and 2) and thank David Bird and Patricia Lam for their helpful
comments on the manuscript. The research in Ljerka Kunst’s and Lacey
Samuels’ laboratories is supported by grants from the Natural Sciences and
Engineering Research Council of Canada.
Papers of particular interest published within the period of review have
been highlighted as:
of special interest
of outstanding interest
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39. Suh MC, Samuels AL, Jetter R, Kunst L, Pollard M, Ohlrogge J,
Beisson F: Cuticular lipid composition, surface structure, and
gene expression in Arabidopsis stem epidermis. Plant Physiol
2005, 139:1649-1665.
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Cuticular wax biosynthesis and export Kunst and Samuels 727
40. DeBono A, Yeats TH, Rose JKC, Bird D, Jetter R, Kunst L,
Samuels L: Arabidopsis LTPG is a
glycosylphosphatidylinositol-anchored lipid transfer protein
required for export of lipids to the plant surface. Plant Cell 2009,
21:1230-1238.
This study demonstrates that a GPI-anchored LTP has a role in the export
of wax components to the cuticle in Arabidopsis stems. Although this
evidence ends a long-standing controversy over whether LTPs act as
carriers of wax components to the plant surface, the plasma membrane
localization of the LTPG leads to many other questions, such as what is
the role of the GPI anchor in LTPG function.
41. Lee SB, Go YS, Bae H, Park JH, Cho SH, Cho HJ, Lee DS,
Park OK, Hwang I, Suh MC: Disruption of
glycosylphosphatidylinositol-anchored lipid transfer protein
gene altered cuticular lipid composition, increased
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plastoglobules, and enhanced susceptibility to infection by
the fungal pathogen Alternaria brassicicola. Plant Physiol
2009, 150:42-54.
42. Hooker TS, Lam P, Zheng H, Kunst L: A core subunit of the RNA processing/degrading exosome specifically influences
cuticular wax biosynthesis in Arabidopsis. Plant Cell 2007,
19:904-913.
The positional cloning of the CER7 gene identified a ribonuclease, a core
subunit of the exosome, as a regulatory protein which controls wax
biosynthesis. The proposed target of the CER7 ribonuclease is an mRNA
specifying a transcriptional repressor of the key wax biosynthetic gene
CER3. In wax producing epidermal cells, the CER7 ribonuclease is
suggested to degrade the target repressor mRNA, thereby boosting
CER3 expression and wax production by the alkane-forming pathway
of wax biosynthesis.
Current Opinion in Plant Biology 2009, 12:721–727