Download Unraveling the complex network of cuticular structure and function

Unraveling the complex network of cuticular structure and function
Christiane Nawrath
A hydrophobic cuticle is deposited at the outermost
extracellular matrix of the epidermis in primary tissues of
terrestrial plants. Besides forming a protective shield against
the environment, the cuticle is potentially involved in several
developmental processes during plant growth. A high degree of
variation in cuticle composition and structure exists between
different plant species and tissues. Lots of progress has been
made recently in understanding the different steps of
biosynthesis, transport, and deposition of cuticular
components. However, the molecular mechanisms that
underlie cuticular function remain largely elusive.
Addresses
University of Lausanne, Department of Plant Molecular Biology,
Biophore Building, UNIL-Sorge, CH-1015 Lausanne, Switzerland
Corresponding author: Nawrath, Christiane ([email protected])
Current Opinion in Plant Biology 2006, 9:281–287
This review comes from a themed issue on
Physiology and metabolism
Edited by Eran Pichersky and Krishna Niyogi
Available online 3rd April 2006
1369-5266/$ – see front matter
# 2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.pbi.2006.03.001
Introduction
The plant cuticle forms a hydrophobic coating that covers
nearly all aboveground parts of terrestrial plants and forms
the interface between plant and environment. Although it
is already known that the cuticle plays an important role
in protecting the plant from water loss, chemicals and
biotic aggressors, many aspects of cuticle biology and
function are still obscure [1]. The scope of this review
is to give an overview of recent findings regarding different aspects of cuticle biosynthesis and to discuss the
potential novel functions of this complex structure.
Structure and composition of the cuticle
Several layers can usually be distinguished in the cuticle
of mature organs: an outermost layer formed by epicuticular waxes and a so-called ‘cuticle proper’ made of a
polymer, i.e. cutin or cutan that is embedded in intracuticular wax. The cuticle proper is connected to the cell
wall via a ‘cuticular layer’ that consists of polymer, wax
and polysaccharides [2]. The thickness of the cuticle
varies widely among different plant species and different
organs of the same plant (0.02–200 mm). Arabidopsis
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leaves, for example, are covered by a cuticle of 25–
30 nm [2,3,4]. During development, the L1 layer of
the late globular stage of the embryo deposits a procuticle.
Therefore, the embryo is already coated by a hydrophobic
layer when it detaches from the endosperm [5]. Mutations
that affect the development of the epidermis may thus
affect cuticle formation ([5–8]; Figure 1).
Cuticular waxes are a mixture of hydrophobic compounds
that are composed predominantly of aliphatic lipids, such
as very long-chain fatty acids (VLCFA) and their derivatives. In addition, waxes might contain other compounds,
such as triterpenoids and phenylpropanoids [1,9,10]. The
epicuticular wax may be deposited as a film or in the form
of crystals. Intracuticular waxes, by contrast, are
embedded in the cuticle polymer mainly forming wellpacked subdomains. A refined sampling method, combining mechanical sampling and solvent extraction, revealed
that the wax load of a leaf might differ not only between
its abaxial and adaxial sides but also between intracuticular and epicuticular waxes [11–13]. In addition, wax
deposition also varies among the different specialized
epidermal cells, such as guard cells and trichomes
[11,14]. Interestingly, triterpenoids, when present in
the wax, have been only found in the intracuticular
wax fraction [13,15].
The three-dimensional structure of the cuticular polymer
is largely unknown. Cutin is a polyester whose monomer
composition can be analyzed by gas chromatography/mass
spectrometry. The predominant aliphatic monomers of
most cutins analyzed are C16 and C18 v-hydroxylated
fatty acids, typically carrying in addition hydroxy- or
epoxy groups in mid-chain positions. Besides aliphatic
monomers, cutin also contains glycerol and small amounts
of phenolic compounds [1,4]. The monomer composition
of the Arabidopsis cutin is different from that which has
been described as typical for cutin because it consists of
high amounts of C16- and C18-dicarboxylic acids
[3,16,17]. In addition, very long chain 2-hydroxy fatty
acids (C18–C28) and VLCFAs have been identified in the
cutin of Arabidopsis [3]. Thus, the polyester composition
of Arabidopsis cutin resembles that of suberin rather than
canonical cutin [3,18]. However, these unusual monomers seem to be incorporated in the same polymer as
typical C16 and C18 monomers: reduction of the typical
monomers in Arabidopsis is compensated for by increased
deposition of dicarboxylic acids [16]. It is worth noting
that four different methods for the depolymerization of
Arabidopsis cutin have been explored and that directly
comparable data are still missing (Table 1). The finding
that dicarboxylic acids are the predominant monomers in
Current Opinion in Plant Biology 2006, 9:281–287
282 Physiology and metabolism
Figure 1
Variation in the ultrastructure of the cuticular membrane of Arabidopsis. Each mutant or transgenic plant is presented next its corresponding
wildtype control. (a) C24 and (b) wax2/yore-yore [39]; (c) Col-0 and (d) cutinase-expressing transgenic plant [36]; (e) Col and (f) bdg [35]; (g) Col
and (h) att1[17]; and (i) Col and (j) ace/hth [26]. Variations in the ultrastructure of the cuticular membrane in different organs and mutants of
Arabidopsis can be seen in form of alterations in the thickness (a,b,g,h), density (a,b,g,h) or disruption of the cuticular membrane (c–f,i,j), and by
the position of electron-opaque material in the extracellular matrix (c–f). The cuticle of stems is shown in (a–d), of leaves in (e–h), and of the
stamen in (I,j). Bars represent 100 nm in (a–d,i,j) and 500 nm in (e–h). Typical features of the cuticular membrane are indicated by an arrow.
Arabidopsis cutin implies that monomers that carry several
hydroxy groups should also be present to allow the formation of a polyester. Glycerol might be such a monomer
[3,16].
Current Opinion in Plant Biology 2006, 9:281–287
A cuticular polymer that is resistant to ester-bond hydrolysis, and often called cutan, is present in varying
amounts. Its composition is little understood [4]. In
Arabidopsis, fewer cutin monomers can be hydrolyzed
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Cuticular structure and function Nawrath 283
Table 1
Major monomers of Arabidopsis (Columbia) shoot polyesters.
BF3
LiAlH4/NaOCH3
Stem cutin
Stem polyester
Leaf polyester
Leaf cutin
(mass %) [17]
(mass %) [19]
(mol %) [16]
(mol %) [16]
(area %) [35]
(area %) [3]
Alkan-1-oic acids
C16
C18 (1)
C18 (2)
–
–
–
12
–
5
2.5
1
1.5
6
17
10
16
5
1
1
<1
1
v-Hydroxyacids
C16 v-OH-Acid
C18 (2) v-OH-Acid
3
–
5
5
5
10
1
7
<1
<1
2
2
a,v-Dicarboxylic acids
C16 Di-acid
C18 Di-acid
C18 (1) Di-acid
C18 (2) Di-acid
49
12
–
1
5
–
5
39
5
2
7
42
9
–
5
38
6
2
4
10
11
3
7
21
2-Hydroxyacids
C24 2-OH-acid
C24 (1) 2-OH-acid
C26 2-OH-acid
–
–
–
–
–
–
–
–
–
–
–
–
18
8
6
5
1
2
Multifunctional aliphatics
C16 9/10,16-Di-OH-acid
7-OH C16 Di-acid
17
11
15
–
14
–
6
–
1
–
1
–
Total number of identified monomers
8
12
14
11
28
33
LiAlH4/LiAlD4
NaOCH3
Leaf polyester
Methanolic-HCl
A direct comparison of the values is impossible because the number of components identified and the units used for their quantification are
different [3,16,17,19,35]. To what extent differences in composition are due to the methodology, data interpretation or to the
environmental conditions in which the plants were grown will have to be investigated further. Only monomers that contribute more than 5% to
the polyester in at least one preparation are represented.
from older stem sections than from young ones, indicating
that cutin might be cross-linked by non-ester bonds after
its primary deposition [19].
Thus, both the polymer and the wax fraction of the
cuticle show a remarkable variability in composition
and structure.
Biosynthesis of aliphatic cuticle components
Aliphatic wax molecules are synthesized by the fatty acid
elongase (FAE) from C16 and C18 fatty acids, resulting in
VLCFAs ranging from C24 to C36 in length. VLCFAs
enter either the acyl reduction pathway, leading to the
formation of primary alcohols and wax esters, or the
decarbonylation pathway, which synthesizes aldehydes,
alkanes, secondary alcohols and ketones [9,20]. The
biosynthesis of VLCFA is beginning to be unraveled,
but the genes that are involved in VLCFA modification
have not yet been identified [9]. ECERIFERUM6
(CER6) is the most important condensing enzyme of
the FAE for cuticular wax biosynthesis in Arabidopsis,
and is able to elongate C22 and longer fatty-acid-CoAs
[21]. The inflorescence stems of Cer6-sense-suppressed
plants have less than 10% of the wax load of those of
wildtype plants [22]. The cer10 mutant has a 60% reduction in wax load and is defective in the ECR gene, which
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encodes an enoyl-CoA reductase that is required for
VLCFA biosynthesis [23].
Cutin monomers are synthesized by the cytochrome
P450- and/or a peroxygenase-dependent pathway
[4,24]. In Arabidopsis, as identified by the phenotype of
lacerata (lcr) and aberrant induction of type III (att1)
mutants, the cytochrome P450s CYP86A8 and CYP86A2
are involved in cuticle formation [17,25]. att1 is the first
Arabidopsis mutant for which both an altered ultrastructure of the cuticular membrane and an altered cutin
monomer composition have been described. att1 has a
cuticle that is less osmophilic but is more than twice as
thick as that of wildtype plants (Figure 1g,h). Most cutin
monomers were strongly reduced in att1/cyp86a (31–78%
less than in wildtype), indicating that ATT1/CYP86A has
a broad function in cutin biosynthesis [17]. Compositional and ultrastructural changes have also been identified in the cuticular polyester of lcr (A Yephremov,
unpublished). Adhesion of calyx edges/hothead (ace/hth)
mutants are characterized by a reduced amount of dicarboxylic acids in the cuticular polyester and a disrupted
cuticular membrane (Figure 1j; [26]). ACE/HTH encodes
an oxidoreductase that most probably catalyzes the oxidation of hydroxylated fatty acids to aldehydes [26,27].
The long-chain fatty acid-CoA synthethase LACS2 of
Current Opinion in Plant Biology 2006, 9:281–287
284 Physiology and metabolism
Arabidopsis is expressed in an epidermis-specific pattern
and has a preference for v-hydroxy fatty acids when
expressed in Escherichia coli [28]. The properties and
appearance of the cuticular membrane are altered in lacs2
mutants, indicating that a specific step of CoA-ester
formation is necessary during cutin biosynthesis [28].
Other genes that are potentially involved in cutin or wax
biosynthesis are FIDDLEHEAD (FDH) and HIGH CARBON DIOXIDE (HIC), both of which encode condensing
enzymes of the CER6 family that are potentially involved
in VLCFA synthesis [29–31]. Additional candidate genes
have been identified by transcriptome analysis of epidermal peels of stem sections [19].
several WBC-homologs might act in wax export as their
expression is strongly enriched in the epidermis of rapidly
elongating stem segments [19]. Different mechanisms
of CER5 action during the secretion of wax molecules
have been discussed recently [32]. Whether cutin monomers use a similar transport system remains to be
elucidated.
Cell biological aspects of wax and cutin
biosynthesis
The formation of cuticular polymers and the organization
of polymers and waxes into a defined extracellular structure are also little understood [1,4]. The first component
that is involved directly in the formation of the cuticular
membrane of Arabidopsis is BODYGUARD (BGD) [35].
BDG encodes an extracellular-localized protein of the
superfamily of a/b hydrolases, the family to which cutinase also belongs. bdg mutants accumulate greater than
wildtype amounts of normally composed hydrolysable
polyester. The structure of the cuticular membrane of
bdg is, however, highly irregular and at places interrupted
or double layered, with large amounts of the polyester
located within the cell wall (Figure 1f; [35]). Therefore,
a synthase activity has been proposed for BDG as
reported for other members of this superfamily. A certain
similarity between the ultrastructure of the cuticles of bdg
leaves and the inflorescence stems of cutinase-expressing
Arabidopsis plants has been found (Figure 1h; [35,36]).
Thus, BDG could have both synthase and hydrolase
activity in cuticle remodeling or homeostasis during plant
growth [35].
The biosynthesis of cuticular components branches off
from the de novo fatty biosynthetic pathway in the plastid.
C16- and C18-fatty acids are liberated by the action of the
acyl-ACP thioesterases (FatA and FatB), exported to the
cytoplasm, and conjugated to CoA. Acyl-ACP thioesterases regulate the chain-length distribution of the
monomers of the cuticular polyester [16]. Acyl-CoAs
might then be channeled to different pathways for wax
and cutin monomer biosynthesis. The elongation and
cytochrome P450-dependent hydroxylation of fatty acids
takes place at the endoplasmic reticulum (ER). VCLFA
might then be channeled directly to the different
enzymes complexes that are necessary for their modification. A mutation in the ER-located FAD2 desaturase also
reduces the degree of desaturation of cutin monomers,
indicating that some of the unsaturated cutin precursors
are taken from phospholipids [16]. Whether cytochrome
P450-dependent hydroxylation steps and other reactions
that are involved in cutin monomer biosynthesis occur on
fatty acids that are attached to phospholipids remains to
be elucidated. The involvement of LACS2 in cutin
biosynthesis indicates that, to some degree, free fatty
acid derivatives are formed as intermediates and then
conjugated to CoA again [28].
Many open questions exist about the different steps in
the transport of cuticular components from the ER to the
plasma membrane, across the plasma membrane and
across the cell wall to the cuticle [9,32]. Recently, one
of the components of wax export across the plasma
membrane has been isolated. The CER5 gene of Arabidopsis encodes WBC12, an ABC-transporter of the
WHITE-BROWN COMPLEX (WBC) subfamily
[33]. A mutation in the CER5 locus leads to a decreased
extracellular wax load but does not change total wax
content, indicating that the unusual sheet-like structures
that are located in the cytoplasmic protrusions into the
vacuole of the epidermal cell might be waxes that could
not be secreted. Thus, CER5 is hypothesized to act
directly in the export of wax molecules, potentially with
a preference for alkanes [33,34]. In addition to CER5,
Current Opinion in Plant Biology 2006, 9:281–287
The transport of highly hydrophobic wax molecules
across the hydrophilic cell wall remains the biggest puzzle
in cuticle biosynthesis. Different hypothesis for this
transport step have been discussed by Kunst and Samuels
[9].
Nothing is known about the reactions that are involved in
the modification of cutin to cutan. A recent analysis of the
transcriptome of epidermal peels identified 600 genes
that are specifically upregulated in the basal segment of
the stem, which was paralleled by a decrease in the
amount of hydrolysable cutin polymer. Genes that are
involved in cutan formation could be among those upregulated genes [19].
Regulation of the biosynthesis of cuticular
components
The quantity of wax does not change in rapidly elongating stems, indicating that the biosynthetic flux into waxes
must be tightly coordinated with surface area expansion
[19]. Genes that potentially encode regulators of wax
biosynthesis have been identified by a visually detectable
reduction in epicuticular wax load in a number of Arabidopsis mutants (e.g. cer1 and cer3) [9]. Interestingly, mutations in homologs of CER1 or WAX2/YORE-YORE in
Arabidopsis and in GLOSSY1 in maize, lead to obvious
alterations in both the cuticular membrane and wax
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Cuticular structure and function Nawrath 285
phenotypes, suggesting a general regulatory function for
cuticular components (Figure 1a,b; [37–39]).
increased permeability of their leaf cuticles, whereas all
other cer mutants do not [46].
Members of the WAX INDUCER (WIN)/SHINE family of
Arabidopsis genes encode transcriptional activators that
potentially act in wax biosynthesis. The overexpression of
these genes leads to an increase in wax load, an overexpression of CER1, and altered properties of the cuticular membrane, suggesting a regulatory function in the
biosynthesis of both cutin and wax [40,41].
Phenotypes of plants that have an altered
cuticular permeability
A high degree of complexity regarding the regulation of
wax biosynthesis was evidenced by a detailed analysis of
the wax composition of 14 different cer double mutants
[34]. None of the classes of wax molecules can be
completely eliminated and clear epistatic relationships
are rarely observed. Additive or synergistic effects are
often found, indicating a high degree of redundancy in
the pathway. Complex mechanisms might also regulate
the amount of polyester deposited by epidermal cells as
alterations in polyester composition, but not in its total
amount, have been detected in Arabidopsis mutants that
are affected in primary lipid metabolism [16].
Functional aspects of the cuticle
The cuticle is involved in several different functions; for
example, it inhibits the uncontrolled permeation of water,
solutes and gases and the deposition of advertive substances, and it protects the plant against UV irradiation,
mechanical damage, phythopathogens and herbivorous
insects [4,10]. Potentially, the cuticle might also be
involved in the generation and distribution of signals in
development and in plant pathogen–interactions. Aspects
that have been recently reviewed will not been discussed
in detail here [1,14,42,43].
The role of the cuticle as a barrier to water loss is likely to
be its primary and most important function as the formation of the cuticle was a critical step for the evolution of
terrestrial plants [1]. Although it restricts diffusion, the
cuticle is permeable to various molecules, a property that
is of agronomical value as it enables spray application of
various compounds, such as herbicides or fertilizers.
Uncharged and charged molecules travel through the
cuticle on different paths, with larger molecules diffusing
more easily through the cuticle when charged [14]. Different studies have shown that both waxes and cutin are
important for the formation of the diffusion barrier [1].
The importance of an intact cuticular ultrastructure in
restricting the permeability of the cuticle to uncharged
molecules was confirmed in several Arabidopsis mutants
and transgenic plants that expressed a fungal cutinase
[17,26,27,28,35,36,39,40,44–46]. The enhanced permeability of a defective cuticle has been used in a forward
genetic approach to identify novel Arabidopsis mutants,
the permeable leaves ( pel) mutants [46]. Interestingly, four
cer mutants, namely cer10, cer12, cer14, and cer19, exhibit
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One of the most striking phenotypes that often correlates
with cuticular defects is the formation of organ fusions, as
observed in fdh, lcr, wax2, bgd, ace/hth and several other
Arabidopsis mutants and in transgenic plants that express
a fungal cutinase [25,29,35,36,39,46,47]. A potential
explanation for the fusion phenotype is that the cuticle
blocks the cell-wall co-polymerization of cells from individual organs.
An argument against this simple explanation might be
that mutants that have cuticular defects exhibit a wide
variety of phenotypes that concern different aspects of
development, such as differences in the form of epidermal pavement cells (in lcr, lacs2, pel1, pel2, cer10 and ace/
hth) and variations in stomata and trichome formation (in
fdh, lcr, wax2/yore-yore, 35S-SHINE and bdg)
[23,25,26,28,29,35,38,40,46,47]. All of these phenotypes could potentially be caused by alterations in the
generation or distribution of signal molecules when the
cuticle is more permeable. The situation has become
even more complex, however, because not all plants that
have permeable cuticles have the same phenotype. For
example, the Arabidopsis mutant lcr/cyp86A8 is an organ
fusion mutant that has an altered trichome development
but att1/cyp86A2 is not [17,25]. By contrast, a mutation in
ATT1 leads to the expression of virulence genes in phytopathogenic bacteria, a phenotype that has not been
found in the wax2/yore-yore mutant [17].
An alternative explanation could be that the synthesis of
cuticular components is interconnected with the generation of lipid-based signals that act in different processes of
development or plant defense [17,23]. For example, the
organ fusion mutant cer10 of Arabidopsis is defective in the
enoyl-CoA reductase ECR. This enzyme is important for
the synthesis of all VLCFAs, which are used for wax,
sphingolipids, storage lipids, and potentially cutin monomers [23]. Similarly, the resurrection mutant revealed a
link between the deposition of waxes, seed lipids, and
embryo development [48].
The number of known cuticle-related phenotypes and
phenomena is still increasing. For example, a strong
resistance of cutinase-expressing Arabidopsis plants to
the necrotrophic fungus Botrytis cinerea has recently been
reported [43]. Moreover, the potential of certain epiphytic
bacteria to alter cuticle permeability has been described
[49]. Also, the success of Erysiphe pisi in infesting pea
plants might depend on the composition of the epicuticular wax layer [11]. Thus, the elucidation of a potential
role of the cuticle in plant development and plant–pathogen interactions will remain a hot topic in the future.
Current Opinion in Plant Biology 2006, 9:281–287
286 Physiology and metabolism
Arabidopsis thaliana, that is expressed in the outer cell layers
of embryos and plants, is involved in proper embryogenesis.
Plant Cell Physiol 2002, 43:419-428.
Conclusions
The combination of recent advances in the chemical
analysis of cuticular components with forward and reverse
genetic approaches is well under way [18,19]. A large
number of genes that are involved in cutin and wax
biosynthesis and their regulation will thus be identified
soon. The relation between the structure and function of
the cuticle at the molecular level is still unknown, however, and the physical properties of cutin have only been
thoroughly studied for tomato [50]. Several different
characteristic ultrastructural features of the cuticular
membrane of Arabidopsis have now been identified
(Figure 1), and diverse phenotypes of mutants that have
alterations in their cuticle have also been recognized.
Both types of information might help to unravel important relationships between composition, structure, and
function. However, knowledge from Arabidopsis needs to
be complemented with information from other plant
systems because the Arabidopsis cuticle is ultra-thin
and of uncommon composition. Refined chemical analysis methods will have to be developed to answer questions
regarding tissue- and cell-specific composition, microstructure, and function. The diversity of the structure
and composition of the cuticle might, however, already
give us a clue to the diversity of functional mechanisms in
which the cuticle is involved.
Acknowledgements
I would like to thank Gustavo Bonaventure, Rochus Franke, Alexander
Yephremov, and Simon Goepfert for critical reading of the manuscript
and helpful discussions. The research in CN’s laboratory is funded
by the Swiss National Foundation (grant #3100A0-109405/1).
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
of special interest
of outstanding interest
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Cuticular structure and function Nawrath 287
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