Constructing functional cuticles: analysis of relationships between cuticle lipid composition, ultrastructure and water barrier function in developing adult maize leaves

Background and Aims Prior work has examined cuticle function, composition and ultrastructure in many plant species, but much remains to be learned about how these features are related. This study aims to elucidate relationships between these features via analysis of cuticle development in adult maize (Zea mays L.) leaves, while also providing the most comprehensive investigation to date of the composition and ultrastructure of adult leaf cuticles in this important crop plant. Methods We examined water permeability, wax and cutin composition via gas chromatography, and ultrastructure via transmission electron microscopy, along the developmental gradient of partially expanded adult maize leaves, and analysed the relationships between these features. Key Results The water barrier property of the adult maize leaf cuticle is acquired at the cessation of cell expansion. Wax types and chain lengths accumulate asynchronously over the course of development, while overall wax load does not vary. Cutin begins to accumulate prior to establishment of the water barrier and continues thereafter. Ultrastructurally, pavement cell cuticles consist of an epicuticular layer, and a thin cuticle proper that acquires an inner, osmiophilic layer during development. Conclusions Cuticular waxes of the adult maize leaf are dominated by alkanes and alkyl esters. Unexpectedly, these are localized mainly in the epicuticular layer. Establishment of the water barrier during development coincides with a switch from alkanes to esters as the major wax type, and the emergence of an osmiophilic (likely cutin-rich) layer of the cuticle proper. Thus, alkyl esters and the deposition of the cutin polyester are implicated as key components of the water barrier property of adult maize leaf cuticles.

Changes in lipid composition and ultrastructure associated with functional maturation of the cuticle during adult maize leaf development

Although extensive prior work has characterized cuticle composition, function, ultrastructure and development in many plant species, much remains to be learned about how these features are interrelated. Moreover, very little is known about the adult maize leaf cuticle in spite of its significance for agronomically important traits in this major crop. We analyzed cuticle composition, ultrastructure, and permeability along the developmental gradient of partially expanded adult maize leaves to probe the relationships between these features. The water barrier property is acquired at the cessation of cell expansion. Wax types and chain lengths accumulate asynchronously along the developmental gradient, while overall wax load does not vary. Cutin begins to accumulate prior to establishment of the water barrier and continues thereafter. Ultrastructurally, pavement cell cuticles consist of an epicuticular layer, a thin cuticle proper that acquires an inner, osmiophilic layer during development, and no cuticular layer. Cuticular waxes of the adult maize leaf are dominated by alkanes and wax esters localized mainly in the epicuticular layer. Establishment of the water barrier coincides with a switch from alkanes to esters as the major wax type, and the emergence of an osmiophilic (likely cutin-rich) layer of the cuticle proper.Higlight statementChemical, ultrastructural and functional analysis of cuticle development in partially expanded adult maize leaves revealed important roles for wax esters and an osmiophilic, likely cutin-rich, layer in protection from dehydration.

Microscopy of crustacean cuticle: formation of a flexible extracellular matrix in moulting sea slaters Ligia pallasii

Structural and functional properties of exoskeleton in moulting sea slaters Ligia pallasii from the Eastern Pacific coast were investigated with CT scanning and electron microscopy. Ultrastructure of preecdysial and postecdysial cuticular layers was described in premoult, intramoult and postmoult animals. Cuticle is a flexible extracellular matrix connected to the epidermal cells through pore channels. During premoult epicuticle and exocuticle are formed and during intramoult and postmoult endocuticular lamellae are deposited and the cuticle is progressively constructed by thickening and mineralization. Cuticle permeability, flexibility and waterproofing capacity change accordingly. Elaboration of epicuticular scales connected to an extensive network of nanotubules, establish its anti-adhesive and hydrophobic properties. Labelling with gold conjugated WGA lectins on Tokuyashu thawed cryosections exposes differences in chitin content between exocuticle and endocuticle. Histochemical staining of cuticle shows presence of acidic carbohydrates/glycoconjugates and lipoproteins in epicuticular layer. Chitin microfibrils are formed at the microvillar border of epidermal cells with abundant Golgi apparatus and secretory vesicles. Numerous spherules associated with nanotubules were observed in the ecdysial space in intramoult animals. The mineral component of the cuticle as visualized with CT scanning indicates progressive mineral resorption from the posterior to the anterior half of the body in premoult animals, its translocation from the anterior to posterior part during intramoult and its progressive deposition in the posterior and anterior exoskeleton during postmoult. Cuticle of sea slaters is a unique biocomposite and biodynamic material constantly reconstructed during frequent moults, and adapted to specific physical and biotic conditions of the high intertidal rocky zone.

Long noncoding miRNA gene represses wheat β-diketone waxes

The cuticle of terrestrial plants functions as a protective barrier against many biotic and abiotic stresses. In wheat and other Triticeae, β-diketone waxes are major components of the epicuticular layer leading to the bluish-white glaucous trait in reproductive-age plants. Glaucousness in durum wheat is controlled by a metabolic gene cluster at the WAX1 (W1) locus and a dominant suppressor INHIBITOR of WAX1 (Iw1) on chromosome 2B. The wheat D subgenome from progenitor Aegilops tauschii contains W2 and Iw2 paralogs on chromosome 2D. Here we identify the Iw1 gene from durum wheat and demonstrate the unique regulatory mechanism by which Iw1 acts to suppress a carboxylesterase-like protein gene, W1-COE, within the W1 multigene locus. Iw1 is a long noncoding RNA (lncRNA) containing an inverted repeat (IR) with >80% identity to W1-COE. The Iw1 transcript forms a miRNA precursor-like long hairpin producing a 21-nt predominant miRNA, miRW1, and smaller numbers of related sRNAs associated with the nonglaucous phenotype. When Iw1 was introduced into glaucous bread wheat, miRW1 accumulated, W1-COE and its paralog W2-COE were down-regulated, and the phenotype was nonglaucous and β-diketone–depleted. The IR region of Iw1 has >94% identity to an IR region on chromosome 2 in Ae. tauschii that also produces miRW1 and lies within the marker-based location of Iw2. We propose the Iw loci arose from an inverted duplication of W1-COE and/or W2-COE in ancestral wheat to form evolutionarily young miRNA genes that act to repress the glaucous trait.

Cytochrome P450 associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae

The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of 14C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An. gambiae.

Effects of above-limit ground-level ozone concentrations on the health condition of the silver birch (Betula pendula Roth) assimilation apparatus within the period of 2009–2011

Effects of above-limit ground-level ozone concentrations on the health condition of the silver birch (Betula pendula Roth) assimilation apparatus was monitored using the method of study of changes in epicuticular waxes. These waxes are present on the surface of leaves and protect the plant (and, especially, its assimilation apparatus) against negative environmental effects. Studies were performed in a tree stand of the 2nd age category in altitudes ranging from 600 to 1,050 m in the Ore Mountains (Krušné hory in Czech). It was found out that, as compared with a control stand, the thickness of the wax epicuticular layer decreased with the increasing altitude while concentrations of the ground-level ozone increased. A visible damage of the assimilation apparatus was observed in localities with the highest concentrations of ground-level ozone. Here, not only an overall decrease in the amount of epicuticular waxes was observed but also a degradation of their structure. A statistical analysis of results proved that the loss of epicuticular waxes was significantly dependent on above-limit concentrations of ground-level ozone.

Viscoelastic nanoscale properties of cuticle contribute to the high-pass properties of spider vibration receptor ( Cupiennius salei Keys)

Atomic force microscopy (AFM) and surface force spectroscopy were applied in live spiders to their joint pad material located distal of the metatarsal lyriform organs, which are highly sensitive vibration sensors. The surface topography of the material is sufficiently smooth to probe the local nanomechanical properties with nanometre elastic deflections. Nanoscale loads were applied in the proximad direction on the distal joint region simulating the natural stimulus situation. The force curves obtained indicate the presence of a soft, liquid-like epicuticular layer (20–40 nm thick) above the pad material, which has much higher stiffness. The Young modulus of the pad material is close to 15 MPa at low frequencies, but increases rapidly with increasing frequencies approximately above 30 Hz to approximately 70 MPa at 112 Hz. The adhesive forces drop sharply by about 40% in the same frequency range. The strong frequency dependence of the elastic modulus indicates the viscoelastic nature of the pad material, its glass transition temperature being close to room temperature (25±2 °C) and, therefore, to its maximized energy absorption from low-frequency mechanical stimuli. These viscoelastic properties of the cuticular pad are suggested to be at least partly responsible for the high-pass characteristics of the vibration sensor's physiological properties demonstrated earlier.

Novel Epicuticular Leaf Flavonoids from Kalmia and Gaidtheria (Ericaceae)

Leaves of a number of Ericaceous plants have a thin epicuticular layer that consists mostly of triterpenes. In Kalmia angustifolia, K. latifolia and K. polifolia as well as in Gaultheria procumbens and G. shallon this material also contains trace amounts of flavonoid aglycones. In Kalmia they are the 3-OMe-derivatives of four C-methyl flavones reported previously as the typical leaf-wax flavonoids of Eucalyptus. The new compounds, 5-OH-3,7,4′-triOMe-6,8-diCH3-flavone (kalmiatin), 5-OH-3,7,4′-triOMe-6-CH3-flavone (8-desmethyl-kalmiatin), 5,4′-diOH-3,7-diOMe-6,8-diCH3-flavone (latifolin) and 5,4′-diOH-3,7-diOMe-6-CH3-flavone (8-desmethyl-latifolin) are novel natural products. They could not be detected in eight species of Eucalyptus. A screening of 15 species of Ericaceae revealed that 8-desmethyl-sideroxylin and 8-desmethyl-latifolin are present also in the epicuticular layer on leaves of Gaultheria procumbens, while on G. shallon and on Andromeda polifolia traces of galangin-3-methyl ether were found. In the other species checked no external flavonoid aglycones could be detected.

Ultrastructure d'une glande sternale tubuleuse des mâles de Speonomus hydrophilus (Coleoptera, Bathysciinae)

The tubular sternal glands of S. hydrophilus are tegumentary glands present only in the males and were until today unknown. They lie in the segments 6–8 and open between the 8th and 9th segment. They consist of a ramified epithelium made up of prismatic cells. The gland opening is composed of a porous plate connected to an internal cuticular complex. The porous plate itself consists of an epicuticular layer perforated by tiny pores, a mesocuticular layer with large pore canals reaching the pores, then a part with cuticular filaments. All around the porous plate, the mesocuticle makes up a cylindrical excrescence directed towards the secretory part, the free edge of which is prolonged by long mesocuticular plates called pseudomembranes; they are thin, parallel, and penetrate each of the gland cavities. Thus the tubular sternal glands appear as a ramified epidermal invagination; only the internal part of the cuticle is accompanying this invagination. The pseudomembranes might play the same role as an end apparatus for the secretory products. These glands may produce a sex pheromone which would allow the female to recognize the male.