ABC-type transporters and cuticle assembly: Linking function to polarity in epidermis cells

The aerial organs of plants are covered with a cuticle, a continuous layer overlaying the outermost cell walls of the epidermis. The cuticle is composed of two major classes of the lipid biopolymers: cutin and waxes, collectively termed cuticular lipids. Biosynthesis and transport of cuticular lipids occur predominantly in the epidermis cells. In the transport pathway, cuticular lipids are exported from their site of biosynthesis in the ER/plastid to the extracellular space through the plasma membrane and cell wall. Growing evidence suggests that ATP-binding cassette (ABC) transporters are implicated in transport of cuticular lipids across the plasma membrane of epidermal cells. The Arabidopsis ABC-type transporter protein CER5 (WBC12) was reported to act as a wax monomers transporter. In recent works, our group and others showed that a CER5-related protein, DESPERADO (DSO/WBC11), is required for cutin and wax monomers transport through the plasma membrane of Arabidopsis epidermis cells. Unlike the cer5 mutant, DSO loss-of-function had a profound effect on plant growth and development, particularly dwarfism, postgenital organ fusions, and altered epidermal cell differentiation. The partially overlapping function of CER5 and DSO and the fact that these proteins are half-size ABC transporters suggest that they might form a hetero-dimeric complex while transporting wax components. An intriguing observation was the polar localization of DSO in the distal part of epidermis cells. This polar expression might be explained by DSO localization within lipid rafts, specific plasma membrane microdomains which are associated with polar protein expression. In this review we suggest possible mechanisms for cuticular lipids transport and a link between DSO function and polar expression. Furthermore, we also discuss the subsequent transport of cuticular constituents through the hydrophobic cell wall and the possible involvement of lipid transfer proteins in this process.Key words: ABC transporter, cuticular lipids, polar expression, plasma membrane, epidermis     

A member of the PLEIOTROPIC DRUG RESISTANCE family of ATP binding cassette transporters is required for the formation of a functional cuticle in Arabidopsis

Although the multilayered structure of the plant cuticle was discovered many years ago, the molecular basis of its formation and the functional relevance of the layers are not understood. Here, we present the permeable cuticle1 (pec1) mutant of Arabidopsis thaliana, which displays features associated with a highly permeable cuticle in several organs. In pec1 flowers, typical cutin monomers, such as ω-hydroxylated fatty acids and 10,16-dihydroxypalmitate, are reduced to 40% of wild-type levels and are accompanied by the appearance of lipidic inclusions within the epidermal cell. The cuticular layer of the cell wall, rather than the cuticle proper, is structurally altered in pec1 petals. Therefore, a significant role for the formation of the diffusion barrier in petals can be attributed to this layer. Thus, pec1 defines a new class of mutants. The phenotypes of the pec1 mutant are caused by the knockout of ATP BINDING CASSETTEG32 (ABCG32), an ABC transporter from the PLEIOTROPIC DRUG RESISTANCE family that is localized at the plasma membrane of epidermal cells in a polar manner toward the surface of the organs. Our results suggest that ABCG32 is involved in the formation of the cuticular layer of the cell wall, most likely by exporting particular cutin precursors from the epidermal cell.     

Cuticular defects lead to full immunity to a major plant pathogen

In addition to its role as a barrier, the cuticle is also a source of signals perceived by invading fungi. Cuticular breakdown products have been shown previously to be potent inducers of cutinase or developmental processes in fungal pathogens. Here the question was addressed as to whether plants themselves can perceive modifications of the cuticle. This was studied using Arabidopsis thaliana plants with altered cuticular structure. The expression of a cell wall-targeted fungal cutinase in A. thaliana was found to provide total immunity to Botrytis cinerea. The response observed in such cutinase-expressing plants is independent of signal transduction pathways involving salicylic acid, ethylene or jasmonic acid. It is accompanied by the release of a fungitoxic activity and increased expression of members of the lipid transfer protein, peroxidase and protein inhibitor gene families that provide resistance when overexpressed in wild-type plants. The same experiments were made in the bodyguard (bdg) mutant of A. thaliana. This mutant exhibits cuticular defects and remained free of symptoms after inoculation with B. cinerea. The expression of resistance was accompanied by the release of a fungitoxic activity and increased expression of the same genes as observed in cutinase-expressing plants. Structural defects of the cuticle can thus be converted into an effective multi-factorial defence, and reveal a hitherto hidden aspect of the innate immune response of plants.     

Unraveling the complex network of cuticular structure and function

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.     

Arabidopsis LTPG Is a Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface

Plant epidermal cells dedicate more than half of their lipid metabolism to the synthesis of cuticular lipids, which seal and protect the plant shoot. The cuticle is made up of a cutin polymer and waxes, diverse hydrophobic compounds including very-long-chain fatty acids and their derivatives. How such hydrophobic compounds are exported to the cuticle, especially through the hydrophilic plant cell wall, is not known. By performing a reverse genetic screen, we have identified LTPG, a glycosylphosphatidylinositol-anchored lipid transfer protein that is highly expressed in the epidermis during cuticle biosynthesis in Arabidopsis thaliana inflorescence stems. Mutant plant lines with decreased LTPG expression had reduced wax load on the stem surface, showing that LTPG is involved either directly or indirectly in cuticular lipid deposition. In vitro 2-p-toluidinonaphthalene-6-sulfonate assays showed that recombinant LTPG has the capacity to bind to this lipid probe. LTPG was primarily localized to the plasma membrane on all faces of stem epidermal cells in the growing regions of inflorescence stems where wax is actively secreted. These data suggest that LTPG may function as a component of the cuticular lipid export machinery.     

Exoskeletal cuticle differentiation during intramarsupial development of Porcellio scaber (Crustacea: Isopoda)

Exoskeletal crustacean cuticle is a calcified apical extracellular matrix of epidermal cells, illustrating the chitin-based organic scaffold for biomineralization. Studies of cuticle formation during molting reveal significant dynamics and complexity of the assembly processes, while cuticle formation during embryogenesis is poorly investigated. This study reveals in the terrestrial isopod Porcellio scaber, the ultrastructural organization of the differentiating precuticular matrices and exoskeletal cuticles during embryonic and larval intramarsupial development. The composition of the epidermal matrices was obtained by WGA lectin labelling and EDXS analysis. At least two precuticular matrices, consisting of loosely arranged material with overlying electron dense lamina, are secreted by the epidermis in the mid-stage embryo. The prehatching embryo is the earliest developmental stage with a cuticular matrix consisting of an epicuticle and a procuticle, displaying WGA binding and forming cuticular scales. In newly hatched marsupial larva manca, a new cuticle is formed and calcium sequestration in the cuticle is evident. Progression of larval development leads to the cuticle thickening, structural differentiation of cuticular layers and prominent cuticle calcification. Morphological characteristics of exoskeleton renewal in marsupial manca are described. Elaborated cuticle in marsupial larvae indicates the importance of the exoskeleton in protection and support of the larval body in the marsupium and during the release of larvae in the external environment.     

Cutinsomes and lipotubuloids appear to participate in cuticle formation in Ornithogalum umbellatum ovary epidermis: EM–immunogold research

The outer wall of Ornithogalum umbellatum ovary and the fruit epidermis are covered with a thick cuticle and contain lipotubuloids incorporating ³H-palmitic acid. This was earlier evidenced by selective autoradiographic labelling of lipotubuloids. After post-incubation in a non-radioactive medium, some marked particles insoluble in organic solvents (similar to cutin matrix) moved to the cuticular layer. Hence, it was hypothesised that lipotubuloids participated in cuticle synthesis. It was previously suggested that cutinsomes, nanoparticles containing polyhydroxy fatty acids, formed the cuticle. Thus, identification of the cutinsomes in O. umbellatum ovary epidermal cells, including lipotubuloids, was undertaken in order to verify the idea of lipotubuloid participation in cuticle synthesis in this species. Electron microscopy and immunogold method with the antibodies recognizing cutinsomes were used to identify these structures. They were mostly found in the outer cell wall, the cuticular layer and the cuticle proper. A lower but still significant degree of labelling was also observed in lipotubuloids, cytoplasm and near plasmalemma of epidermal cells. It seems that cutinsomes are formed in lipotubuloids and then they leave them and move towards the cuticle in epidermal cells of O. umbellatum ovary. Thus, we suggest that (1) cutinsomes could take part in the synthesis of cuticle components also in plant species other than tomato, (2) the lipotubuloids are the cytoplasmic domains connected with cuticle formation and (3) this process proceeds via cutinsomes.     

Studies on the Ultrastructure and Histochemistry of Plant Cuticles: The Cuticular Membrane of Agave americana L. in situ

The outer epidermal wall of Agave americana leaves was examinedin order to gain more information about the location and chemicalconstitution of the structural components. In middle aged leavesthe wall comprised six layers which were designated epicuticularwax, cuticle proper, exterior and interior cuticular layer,exterior and interior cellin wall. A lamellated structure, consistingof a series of electron translucent lamellae of uniform thicknessalternating with opaque ones of variable thickness, was observedin the thin cuticle proper on the outside of the cuticular membrane,even without heavy metal treatment. The cuticular layers underneathformed the bulk of the cuticular membrane and they also hadtwo components, an amorphous matrix permeated by a reticulumof fibrillae. Cutin, detected with osmium and with iodine/iodine-sulphuricacid–silver proteinate, was a major component of the opaquelamellae of the cuticle proper and the matrix of the cuticularlayer. Carbohydrates were absent from the cuticle proper butwere detected specifically in the fibrillae of the cuticularlayer and in the cellin wall. Pectic material seemed to be presenton both sides of the junction between cuticular membrane andcellin wall, but no discrete zone corresponding to light microscopicalobservations was detected in the electron microscope. Althoughthe lucent lamellae of the cuticle proper were tentatively ascribedto wax there was no structural or ultrahistochemical evidencefor the wax component of the cuticular layer. The various ultrahistochemicalreactions are discussed in relation to the known chemical compositionof the membrane. Agave americana L., epidermis wall, cuticular membrane, cuticle proper, cuticular layer, ultrahistochemistry, wax     

Cell specification in the Arabidopsis root epidermis requires the activity of ECTOPIC ROOT HAIR 3--a katanin-p60 protein.

The Arabidopsis root is composed of radial cell layers, each with distinct identities. The epidermal layer is composed of rows of hair cells flanked on either side by rows of non-hair epidermal cells. The development of hair and non-hair cells is dependent on domains of positional information with strict boundaries. The pattern of cell differentiation and the expression of molecular markers of cell fate is altered in the ectopic root hair 3 (erh3) mutant epidermis indicating that ERH3 is required for the specification of cell fates from early in development (in the meristem) through differentiation. Furthermore the expression of molecular markers indicates that the specification of cell identities is defective within other radial cell layers. ERH3 encodes a p60 katanin protein that is expressed throughout the plant. Katanin proteins are known to sever microtubules, and have a role in the organisation of the plant cell wall since mutants with decreased katanin activity have been shown to have defective walls. We suggest that microtubules are involved in the specification of cell identities in cells of the Arabidopsis root. Microtubules may be required for the localization of positional cues in the wall that have previously been shown to operate in the development of the root epidermis. Alternatively microtubules may be involved in another as yet undefined process required for the specification of cell identity in plants.     

Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato

Plant epidermal cells have evolved specialist functions associated with adaptation to stress. These include the synthesis and deposition of specialized metabolites such as waxes and cutin together with flavonoids and anthocyanins, which have important roles in providing a barrier to water loss and protection against UV radiation, respectively. Characterization of the sticky peel (pe) mutant of tomato (Solanum lycopersicum) revealed several phenotypes indicative of a defect in epidermal cell function, including reduced anthocyanin accumulation, a lower density of glandular trichomes, and an associated reduction in trichome-derived terpenes. In addition, pe mutant fruit are glossy and peels have increased elasticity due to a severe reduction in cutin biosynthesis and altered wax deposition. Leaves of the pe mutant are also cutin deficient and the epicuticular waxes contain a lower proportion of long-chain alkanes. Direct measurements of transpiration, together with chlorophyll-leaching assays, indicate increased cuticular permeability of pe leaves. Genetic mapping revealed that the pe locus represents a new allele of CUTIN DEFICIENT2 (CD2), a member of the class IV homeodomain-leucine zipper gene family, previously only associated with cutin deficiency in tomato fruit. CD2 is preferentially expressed in epidermal cells of tomato stems and is a homolog of Arabidopsis (Arabidopsis thaliana) ANTHOCYANINLESS2 (ANL2). Analysis of cuticle composition in leaves of anl2 revealed that cutin accumulates to approximately 60% of the levels observed in wild-type Arabidopsis. Together, these data provide new insight into the role of CD2 and ANL2 in regulating diverse metabolic pathways and in particular, those associated with epidermal cells.     

Arabidopsis Deficient in Cutin Ferulate encodes a transferase required for feruloylation of ω-hydroxy fatty acids in cutin polyester

The cuticle is a complex aliphatic polymeric layer connected to the cell wall and covers surfaces of all aerial plant organs. The cuticle prevents nonstomatal water loss, regulates gas exchange, and acts as a barrier against pathogen infection. The cuticle is synthesized by epidermal cells and predominantly consists of an aliphatic polymer matrix (cutin) and intracuticular and epicuticular waxes. Cutin monomers are primarily C(16) and C(18) unsubstituted, ω-hydroxy, and α,ω-dicarboxylic fatty acids. Phenolics such as ferulate and p-coumarate esters also contribute to a minor extent to the cutin polymer. Here, we present the characterization of a novel acyl-coenzyme A (CoA)-dependent acyl-transferase that is encoded by a gene designated Deficient in Cutin Ferulate (DCF). The DCF protein is responsible for the feruloylation of ω-hydroxy fatty acids incorporated into the cutin polymer of aerial Arabidopsis (Arabidopsis thaliana) organs. The enzyme specifically transfers hydroxycinnamic acids using ω-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs, preferentially feruloyl-CoA and sinapoyl-CoA, as acyl donors in vitro. Arabidopsis mutant lines carrying DCF loss-of-function alleles are devoid of rosette leaf cutin ferulate and exhibit a 50% reduction in ferulic acid content in stem insoluble residues. DCF is specifically expressed in the epidermis throughout all green Arabidopsis organs. The DCF protein localizes to the cytosol, suggesting that the feruloylation of cutin monomers takes place in the cytoplasm.     

Development of the crystalliferous cuticle of Chamaecyparis lawsoniana (A. Murr.) Pari. (Gupressaceae)

A developmental study of the cuticle has shown that it consists of a homogeneous cuticle proper apposed on the wall and a heterogeneous cuticular layer generated by intussusception of cutin into the wall. At an early stage, the adcrusted cuticle proper is underlain by a ruthenium red-positive layer in which the cuticular layer originates. The origin of the anticlinal flange is referable to an electron-dense, ruthenium red-positive ridge which arises above the anticlinal wall and which also becomes cutinized. At leaf maturity, the inner surface of the cuticular layer, including that of the flange, forms interdigitating protuberances with the cell wall.
Development of the cuticle coincides with deposition of crystals of calcium oxalate in the epidermal cell wall. Initiation of large, early-formed crystals is associated with electron-opaque membranous structures formed close and parallel to the plasmalemma in the young cell wall. Crystals undergo periclinal and anticlinal growth and subsequently become engulfed within the cuticle by development of the cuticular layer. Cutin/polysaccharide interaction during development and the significance of crystal deposition are discussed.     

Epidermal cell interactions: a case for local talk

Epidermal cell interactions play an important role in reproduction, creating variation in floral structure and modulating gametophyte selection and growth during pollination. Recent progress in understanding the processes by which epidermal cells become responsive to contact, indicate that the developmental quiescence of the epidermis is regulated by a set of genes, some of which modulate the permeability properties of the cell wall and cuticle. Research on mutants of Arabidopsis has revealed a mechanistic uniformity between two processes involving epidermis-mediated interactions that had hitherto been considered to be biologically distinct: postgenital organ fusion and pollen hydration.     

Cell Geometry Guides the Dynamic Targeting of Apoplastic GPI-Linked Lipid Transfer Protein to Cell Wall Elements and Cell Borders in Arabidopsis thaliana

During cellular morphogenesis, changes in cell shape and cell junction topology are fundamental to normal tissue and organ development. Here we show that apoplastic Glycophosphatidylinositol (GPI)-anchored Lipid Transfer Protein (LTPG) is excluded from cell junctions and flat wall regions, and passively accumulates around their borders in the epidermal cells of Arabidopsis thaliana. Beginning with intense accumulation beneath highly curved cell junction borders, this enrichment is gradually lost as cells become more bulbous during their differentiation. In fully mature epidermal cells, YFP-LTPG often shows a fibrous cellulose microfibril-like pattern within the bulging outer faces. Physical contact between a flat glass surface and bulbous cell surface induces rapid and reversible evacuation from contact sites and accumulation to the curved wall regions surrounding the contact borders. Thus, LTPG distribution is dynamic, responding to changes in cell shape and wall curvature during cell growth and differentiation. We hypothesize that this geometry-based mechanism guides wax-carrying LTPG to functional sites, where it may act to “seal” the vulnerable border surrounding cell-cell junctions and assist in cell wall fortification and cuticular wax deposition.     

RDR1 and SGS3, Components of RNA-Mediated Gene Silencing, Are Required for the Regulation of Cuticular Wax Biosynthesis in Developing Inflorescence Stems of Arabidopsis

The cuticle is a protective layer that coats the primary aerial surfaces of land plants and mediates plant interactions with the environment. It is synthesized by epidermal cells and is composed of a cutin polyester matrix that is embedded and covered with cuticular waxes. Recently, we have discovered a novel regulatory mechanism of cuticular wax biosynthesis that involves the ECERIFERUM7 (CER7) ribonuclease, a core subunit of the exosome. We hypothesized that at the onset of wax production, the CER7 ribonuclease degrades an mRNA specifying a repressor of CER3, a wax biosynthetic gene whose protein product is required for wax formation via the decarbonylation pathway. In the absence of this repressor, CER3 is expressed, leading to wax production. To identify the putative repressor of CER3 and to unravel the mechanism of CER7-mediated regulation of wax production, we performed a screen for suppressors of the cer7 mutant. Our screen resulted in the isolation of components of the RNA-silencing machinery, RNA-DEPENDENT RNA POLYMERASE1 and SUPPRESSOR OF GENE SILENCING3, implicating RNA silencing in the control of cuticular wax deposition during inflorescence stem development in Arabidopsis (Arabidopsis thaliana).     

Isolation and characterization of a cDNA encoding a membrane bound acyl-CoA binding protein from Agave americana L. epidermis.

A cDNA encoding an acyl-CoA binding protein (ACBP) homologue has been cloned from a cDNA library made from mRNA isolated from epidermis of young leaves of Agave americana L. The derived amino acid sequence reveals a protein corresponding to the membrane-associated form of ACBPs only previously described in Arabidopsis and rice. Northern blot analysis showed that the A. americana ACBP gene is mainly expressed in the epidermis of mature zone of the leaves. The epidermis of A. americana leaves have a well developed cuticle with the highest amounts of the cuticular components waxes, cutin and cutan suggesting a potential role of the protein in cuticle formation.