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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 www.sciencedirect.com
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 www.sciencedirect.com
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 www.sciencedirect.com
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 www.sciencedirect.com
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 www.sciencedirect.com
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
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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 1.
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28. Schnurr J, Shockey J, Browse J: The acyl-CoA synthetase encoded by LACS is essential for normal cuticle development in Arabidopsis. Plant Cell 2004, 16:629-642. The paper provides evidence for the involvement of a long-chain fatty acid-CoA synthetase in the formation of the cuticular membrane in Arabidopsis.
41. Aharoni A, Dixit S, Jetter R, Thoenes E, van Arkel G, Pereira A: The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis. Plant Cell 2004, 16:2463-2480.
29. Yephremov A, Wisman E, Huijser P, Huijser C, Wellesen K, Saedler H: Characterization of the FIDDLEHEAD gene of Arabidopsis reveals a link between adhesion response and cell differentiation in the epidermis. Plant Cell 1999, 11:21872201. 30. Pruitt RE, Vielle-Calzada JP, Ploense SE, Grossniklaus U, Lolle SJ: FIDDLEHEAD, a gene required to suppress epidermal cell interactions in Arabidopsis, encodes a putative lipid biosynthetic enzyme. Proc Natl Acad Sci USA 2000, 97:1311-1316. 31. Gray JE, Holroyd GH, van der Lee FM, Bahrami AR, Sijmons PC, Woodward FI, Schuch W, Hetherington AM: The HIC signalling pathway links CO2 perception to stomatal development. Nature 2000, 408:713-716. 32. Schulz B, Frommer WB: A plant ABC transporter takes the lotus seat. Science 2004, 306:622-625. 33. Pighin JA, Zheng HQ, Balakshin LJ, Goodman IP, Western TL, Jetter R, Kunst L, Samuels AL: Plant cuticular lipid export requires an ABC transporter. Science 2004, 306:702-704. This paper describes the cloning of CER5, which encodes an ABCtransporter of the WBC class. The authors present evidence that this transporter might be directly involved in the transport of cuticular wax molecules through the plasma membrane. An important step in wax biosynthesis has thus been identified. 34. Goodwin SM, Rashotte AM, Rahman M, Feldmann KA, Jenks MA: Wax constituents on the inflorescence stems of double eceriferum mutants in Arabidopsis reveal complex gene interactions. Phytochemistry 2005, 66:771-780.
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42. Bird SM, Gray JE: Signals from the cuticle affect epidermal cell differentiation. New Phytol 2003, 157:9-23. 43. Chassot C, Metraux JP: The cuticle as source of signals for plant defense. Plant Biosyst 2005, 139:28-31. 44. Lolle SJ, Cheung AY, Sussex IM: Fiddlehead — an Arabidopsis mutant constitutively expressing an organ fusion program that involves interactions between epidermal cells. Dev Biol 1992, 152:383-392. 45. Lolle SJ, Hsu W, Pruitt RE: Genetic analysis of organ fusion in Arabidopsis thaliana. Genetics 1998, 149:607-619. 46. Tanaka T, Tanaka H, Machida C, Watanabe M, Machida Y: A new method for rapid visualization of defects in leaf cuticle reveals five intrinsic patterns of surface defects in Arabidopsis. Plant J 2004, 37:139-146. 47. Lolle SJ, Pruitt RE: Epidermal cell interactions: a case for local talk. Trends Plant Sci 1999, 4:14-20. 48. Chen X, Goodwin SM, Liu XL, Chen XL, Bressan RA, Jenks MA: Mutation of the RESURRECTION1 locus of Arabidopsis reveals an association of cuticular wax with embryo development. Plant Physiol 2005, 139:909-919. 49. Schreiber L, Krimm U, Knoll D, Sayed M, Auling G, Kroppenstedt RM: Plant–microbe interactions: identification of epiphytic bacteria and their ability to alter leaf surface permeability. New Phytol 2005, 166:589-594. 50. Heredia A: Biophysical and biochemical characteristics of cutin, a plant barrier biopolymer. Biochim Biophys Acta 2003, 1620:1-7.
Current Opinion in Plant Biology 2006, 9:281–287
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 www.sciencedirect.com
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 www.sciencedirect.com
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 www.sciencedirect.com
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 www.sciencedirect.com
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 www.sciencedirect.com
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 1.
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Franke R, Briesen I, Wojciechowski T, Faust A, Yephremov A, Nawrath C, Schreiber L: Apoplastic polyesters in Arabidopsis surface tissues — a typical suberin and a particular cutin. Phytochemistry 2005, 66:2643-2658. Findings presented by Bonaventure et al. [16] are confirmed and complemented by the work reported in this paper, which shows that dicarboxylic acids really are part of the cuticular membrane. In addition, 2-hydroxy acids were identified as another unusual class of monomers that are present in Arabidopsis cutin. 4.
Nawrath C: The biopolymers cutin and suberin. In The Arabidopsis Book. Edited by Somerville CR, Meyerowitz ME. American Society of Plant Biologists; 2002.
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10. Jenks MA, Eigenbrode SD, Lemieux B: Cuticular waxes of Arabidopsis. In The Arabidopsis Book. Edited by Somerville CR, Meyerowitz ME. American Society of Plant Biologists; 2002. 11. Gniwotta F, Vogg G, Gartmann V, Carver TLW, Riederer M, Jetter R: What do microbes encounter at the plant surface? Chemical composition of pea leaf cuticular waxes. Plant Physiol 2005, 139:519-530. 12. Jetter R, Schaffer S, Riederer M: Leaf cuticular waxes are arranged in chemically and mechanically distinct layers: evidence from Prunus laurocerasus L. Plant Cell Environ 2000, 23:619-628. 13. Vogg G, Fischer S, Leide J, Emmanuel E, Jetter R, Levy AA, Riederer M: Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid beta-ketoacyl-CoA synthase. J Exp Bot 2004, 55:1401-1410. 14. Schreiber L: Polar paths of diffusion across plant cuticles: new evidence for an old hypothesis. Ann Bot 2005, 95:1069-1073. 15. Jetter R, Schaffer S: Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes of the epicuticular wax film during leaf development. Plant Physiol 2001, 126:1725-1737. 16. Bonaventure G, Beisson F, Ohlrogge J, Pollard M: Analysis of the aliphatic monomer composition of polyesters associated with Arabidopsis epidermis: occurrence of octadeca-cis-6, cis-9diene-1,18-dioate as the major component. Plant J 2004, 40:920-930. The authors show, by several methods, that the monomer composition of epidermal polyesters is very unusual in Arabidopsis. The influence of certain steps in basic lipid metabolism on polyester composition identifies new components of the cutin biosynthetic pathway. Polyester formation in Arabidopsis is discussed. 17. Xiao F, Goodwin SM, Xiao YM, Sun ZY, Baker D, Tang XY, Jenks MA, Zhou JM: Arabidopsis CYP86A2 represses Pseudomonas syringae type III genes and is required for cuticle development. EMBO J 2004, 23:2903-2913. The cloning of the ATT1 gene identifies cytochrome P450 CYP86A2, which is involved in cuticle formation. CYP86A2 is also important for the repression of virulence genes in bacteria. The mechanistic basis that underlies this phenotype can only be hypothesized. An interesting aspect of the paper is that the att1 mutant does not have any of the developmental defects that are present in other mutants in which the formation of the cuticular membrane is affected. 18. Yephremov A, Schreiber L: The dark side of the cell wall: molecular genetics of plant cuticle. Plant Biosyst 2005, 139:74-79. 19. Suh MC, Samuels AL, Jetter R, Kunst L, Pollard M, Ohlrogge J, Reisson F: Cuticular lipid composition, surface structure, and gene expression in Arabidopsis stem epidermis. Plant Physiol 2005, 139:1649-1665. Epidermal cell expansion is studied in parallel to both wax and cutin deposition and a genome-wide analysis of gene expression during the growth and development of the epidermis. This paper gives a wealth of essential information about cuticle formation in Arabidopsis. 20. Jenks MA, Tuttle HA, Eigenbrode SD, Feldmann KA: Leaf epicuticular waxes of the eceriferum mutants in Arabidopsis. Plant Physiol 1995, 108:369-377. www.sciencedirect.com
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21. Millar AA, Clemens S, Zachgo S, Giblin EM, Taylor DC, Kunst L: CUT1, an Arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-longchain fatty acid condensing enzyme. Plant Cell 1999, 11:825-838. 22. Hooker TS, Millar AA, Kunst L: Significance of the expression of the CER6 condensing enzyme for cuticular wax production in Arabidopsis. Plant Physiol 2002, 129:1568-1580. 23. Zheng H, Rowland O, Kunst L: Disruptions of the Arabidopsis enoyl-CoA reductase gene reveal an essential role for verylong-chain fatty acid synthesis in cell expansion during plant morphogenesis. Plant Cell 2005, 17:1467-1481. 24. Lequeu J, Fauconnier ML, Chammai A, Bronner R, Blee E: Formation of plant cuticle: evidence for the occurrence of the peroxygenase pathway. Plant J 2003, 36:155-164. 25. Wellesen K, Durst F, Pinot F, Benveniste I, Nettesheim K, Wisman E, Steiner-Lange S, Saedler H, Yephremov A: Functional analysis of the LACERATA gene of Arabidopsis provides evidence for different roles of fatty acid omegahydroxylation in development. Proc Natl Acad Sci USA 2001, 98:9694-9699. 26. Kurdyukov S, Faust A, Trenkamp S, Ba¨r S, Franke R, Efremova N, Tietjen K, Schreiber L, Saedler H, Yephremov A: Genetic and biochemical evidence for involvement of a,v-dicarboxylic acids in the formation of extracellular matrix. Planta 2006, in press. The analysis of the polyester monomer composition of the ace/hth mutant identifies a function of the oxidoreductase, namely the conversion of vhydroxyacids to fatty aldehydes. These fatty aldehydes are essential precursors for the formation of the dicarboxylic acids present in the Arabidopsis polyester. The authors also provide evidence that the dicarboxylic acids in the Arabidopsis cuticle are of functional importance.
The analysis of 14 cer double mutants reveals an unexpected complexity in the wax biosynthetic pathway as clear epistasis seems to be the exception. 35. Kurdyukov S, Faust A, Nawrath C, Ba¨r S, Voisin D, Franke R, Schreiber L, Saedler H, Me´traux J-P, Yephremov A: The epidermis-specific extracellular a/b hydrolase BODYGUARD, an Arabidopsis homologue of fungal cutinases, controls cuticle development and morphogenesis in plants. Plant Cell 2006, 18:321-339. BDG is the first extracellular protein identified that is involved in the formation of an intact cuticular membrane. The many similarities between the phenotypes of bdg and cutinase-expressing plants are interesting. The potential functions of BDG as a synthase and/or hydrolase are discussed. 36. Sieber P, Schorderet M, Ryser U, Buchala A, Kolattukudy P, Metraux JP, Nawrath C: Transgenic Arabidopsis plants expressing a fungal cutinase show alterations in the structure and properties of the cuticle and postgenital organ fusions. Plant Cell 2000, 12:721-737. 37. Sturaro M, Hartings H, Schmelzer E, Velasco R, Salamini F, Motto M: Cloning and characterization of GLOSSY1, a maize gene involved in cuticle membrane and wax production. Plant Physiol 2005, 138:478-489. 38. Kurata T, Kawabata-Awai C, Sakuradani E, Shimizu S, Okada K, Wada T: The YORE-YORE gene regulates multiple aspects of epidermal cell differentiation in Arabidopsis. Plant J 2003, 36:55-66. 39. Chen X, Goodwin SM, Boroff VL, Liu XL, Jenks MA: Cloning and characterization of the WAX2 gene of Arabidopsis involved in cuticle membrane and wax production. Plant Cell 2003, 15:1170-1185.
27. Krolikowski KA, Victor JL, Wagler TN, Lolle SJ, Pruitt RE: Isolation and characterization of the Arabidopsis organ fusion gene HOTHEAD. Plant J 2003, 35:501-511.
40. Broun P, Poindexter P, Osborne E, Jiang CZ, Riechmann JL: WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis. Proc Natl Acad Sci USA 2004, 101:4706-4711.
28. Schnurr J, Shockey J, Browse J: The acyl-CoA synthetase encoded by LACS is essential for normal cuticle development in Arabidopsis. Plant Cell 2004, 16:629-642. The paper provides evidence for the involvement of a long-chain fatty acid-CoA synthetase in the formation of the cuticular membrane in Arabidopsis.
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29. Yephremov A, Wisman E, Huijser P, Huijser C, Wellesen K, Saedler H: Characterization of the FIDDLEHEAD gene of Arabidopsis reveals a link between adhesion response and cell differentiation in the epidermis. Plant Cell 1999, 11:21872201. 30. Pruitt RE, Vielle-Calzada JP, Ploense SE, Grossniklaus U, Lolle SJ: FIDDLEHEAD, a gene required to suppress epidermal cell interactions in Arabidopsis, encodes a putative lipid biosynthetic enzyme. Proc Natl Acad Sci USA 2000, 97:1311-1316. 31. Gray JE, Holroyd GH, van der Lee FM, Bahrami AR, Sijmons PC, Woodward FI, Schuch W, Hetherington AM: The HIC signalling pathway links CO2 perception to stomatal development. Nature 2000, 408:713-716. 32. Schulz B, Frommer WB: A plant ABC transporter takes the lotus seat. Science 2004, 306:622-625. 33. Pighin JA, Zheng HQ, Balakshin LJ, Goodman IP, Western TL, Jetter R, Kunst L, Samuels AL: Plant cuticular lipid export requires an ABC transporter. Science 2004, 306:702-704. This paper describes the cloning of CER5, which encodes an ABCtransporter of the WBC class. The authors present evidence that this transporter might be directly involved in the transport of cuticular wax molecules through the plasma membrane. An important step in wax biosynthesis has thus been identified. 34. Goodwin SM, Rashotte AM, Rahman M, Feldmann KA, Jenks MA: Wax constituents on the inflorescence stems of double eceriferum mutants in Arabidopsis reveal complex gene interactions. Phytochemistry 2005, 66:771-780.
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