Cuticular wax biosynthesis in petunia petals: cloning and characterization of an alcohol-acyltransferase that synthesizes wax-esters

The surface of plants is covered by cuticular wax, which contains a mixture of very long-chain fatty acid (VLCFA) derivatives. This wax surface provides a hydrophobic barrier which reduces non-stomatal water loss. One component of the cuticular wax is the alkyl esters, which typically contain a VLCFA esterified to an alcohol of a similar length. As part of an EST project, we recently identified an acyltransferase with 19% sequence identity (amino acid) to a bacterial ‘bifunctional’ wax-ester synthase/diacylglycerol acyltransferase (WS/DGAT). Northern analysis revealed that this petunia homologue was expressed predominantly within the petals. The cDNA encoding the WS/DGAT homologue was introduced into a yeast strain deficient in triacylglycerol biosynthesis. The expressed protein failed to restore triacylglycerol biosynthesis, indicating that it lacked DGAT activity. However, isoamyl esters of fatty acids were detected, which suggested that the petunia cDNA encoded a wax-synthase. Waxes were extracted from petunia petals and leaves. The petal wax extract was rich in VLCFA esters of methyl, isoamyl, and short-to-medium straight chain alcohols (C4–C12). These low molecular weight wax-esters were not present in leaf wax. In-vitro enzymes assays were performed using the heterologously expressed protein and ¹⁴C-labelled substrates. The expressed protein was membrane bound, and displayed a preference for medium chain alcohols and saturated very long-chain acyl-CoAs. In fact, the activity would be sufficient to produce most of the low molecular wax-esters present in petals, with methyl-esters being the exception. This work is the first characterization of a eukaryotic protein from the WS/DGAT family. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Insect wax secretion the growth of tubular crystals

Filaments of a pure long-chain wax secreted by the wooly alder aphid are shown to represent a previously-uncharacterized, tubular, crystal habit for paraffins. Although this habit is never seen in solution or epitaxial growth of such materials, it requires only a circular closure at the wax secretion site, further growth behavior being controlled totally by van der Waal's intermolecular interactions. The linear paraffin-like wax molecules pack in the filament monolayer envelope with chain axes oriented radially from the filament axis. Elongation of the wax filaments corresponds to the strong interactions along the [110] vectors in the O_⊥ polymethylene chain packing, and stabilization of the filament occurs via strong interchain interactions along $\text{[1}\text{1}\text{0]}$ and [010] in helical paths of opposite chirality.     

Sclerotin and lipid in the waterproofing of the insect cuticle

In all the cuticles studied waterproofing is effected by extracuticular material, a mixture of sclerotin precursors and lipids, exuded from the tubular filaments of the pore canals. In Rhodnius larval abdomen it is a layer of thickness similar to the outer epicuticle, believed to be composed of 'sclerotin' and wax, in Schistocerca larval sternal cuticle and in Carausius sternal cuticle it is similar. In Tenebrio adult sternal cuticle of the abdomen, in both the extracuticular exudation and the contents of the distal endings of the tubular filaments, the wax component is obscured by hard 'sclerotin'. In Manduca larva a very thin layer of 'sclerotin' and wax is covered by an irregular wax layer, average 0.75 micron, twice the thickness of the inner epicuticle. In Periplaneta and Blattella the abdominal cuticle is covered by a soft waxy layer, often about 1 micron thick, which is mixed with argentaffin material. Below this is a very thin waterproof layer of wax and 'sclerotin' continuous with the contents of the tubular filaments, which is readily removed by adsorptive dusts. In Apis adult abdominal terga free wax plus sclerotin precursors form a thin layer which is known to be removed by adsorptive dusts. In Calliphora larva there is a very thin layer of the usual mixed wax and sclerotin and below this a thick (0.5 micron) layer, lipid staining and strongly osmiophil, likewise extracuticular and exuded from the epicuticular channels. This material (which is often called 'outer epicuticle') has the same staining and resistance properties as the true outer epicuticle on which it rests. In the abdomen of Calliphora adult the waterproofing wax-sclerotin mixture forms a thin layer over the entire cuticle including the surface of the microtrichia. There is also a thin detachable layer of free wax on the surface.     

Organization of the cuticle of an aquatic fly larva

The aquatic, apneustic larva of the midge, Chironomus riparius, has a very thin (up to 5 micrometers), readily deformable, post-cephalic cuticle. The ultrastructure of this cuticle from newly moulted and older final instar animals, and exuvia shed at pupation, has been examined using routine methods and also after the extraction of proteins with formamide and acetic acid. From the results described, and using established criteria, it is inferred that an exocuticle is present and represents about 25% of the thickness of the mature procuticle, the remainder being endocuticle. Therefore, it would seem that this exceptionally delicate cuticle conforms to the conventional plan of tanned or sclerotized solid cuticles, unlike those reported in the larvae of cyclorrhaphous dipterans such as blowflies or other soft-bodied insects. This is the first account, using experimental techniques, of the fine structure of sectioned cuticle from nematocerous dipteran larvae. It also indicates the value of the exuvium as a source of information about cuticle structure.     

The tanning of insect cuticle—A critical review and a revised mechanism

Two mechanisms to account for the stiffening of cuticle at tanning were proposed in 1940. The quinone tanning theory has been almost universally accepted; that of dehydration almost universally neglected. Calculations and tests on the mechanical properties of cuticle under differing conditions suggest that covalent cross-linking, even if it exists, is insufficient to account for the degree of stiffening of cuticle at sclerotisation. Dehydration will induce sufficient secondary bonds to account for the stiffness and insolubility of ‘tanned’ cuticle in the complete absence of covalent cross-links. It is suggested that the mechanism of sclerotisation is driven by quinones and other chemicals which are secreted into the cuticle at sclerotisation and cause highly controlled dehydration. Covalent cross-linking may still occur, but must be considered to be of secondary importance and unproven in all cuticles other than resilin.     

Composition of the cuticle of developing sweet cherry fruit

The composition of wax and cutin from developing sweet cherry (Prunus avium) fruit was studied by GC-MS between 22 and 85 days after full bloom (DAFB). In this and our previous study, fruit mass and surface area increased in a sigmoidal pattern with time, but mass of the cuticular membrane (CM) per unit fruit surface area decreased. On a whole fruit basis, mass of CM increased up to 36 DAFB and remained constant thereafter. At maturity, triterpenes, alkanes and alcohols accounted for 75.6%, 19.1% and 1.2% of total wax, respectively. The most abundant constituents were the triterpenes ursolic (60.0%) and oleanolic acid (7.5%), the alkanes nonacosane (13.0%) and heptacosane (3.0%), and the secondary alcohol nonacosan-10-ol (1.1%). In developing fruit triterpenes per unit area decreased, but alkanes and alcohols remained essentially constant. The cutin fraction of mature fruit consisted of mostly C16 (69.5%) and, to a lower extent, C18 monomers (19.4%) comprising alkanoic, omega-hydroxyacids, alpha,omega-dicarboxylic and midchain hydroxylated acids. The most abundant constituents were 9(10),16-dihydroxy-hexadecanoic acid (53.6%) and 9,10,18-trihydroxy-octadecanoic acid (7.8%). Amounts of C16 and C18 monomers per unit area decreased in developing fruit, but remained approximately constant on a whole fruit basis. Within both classes of monomers, opposing changes occurred. Amounts of hexadecandioic, 16-hydroxy-hexadecanoic, 9(10)-hydroxy-hexadecane-1,16-dioic and 9,10-epoxy-octadecane-1,18-dioic acids increased, but 9,10,18-trihydroxy-octadecanoic and 9,10,18-trihydroxy-octadecenoic acids decreased. There were no qualitative and minor quantitative differences in wax and cutin composition between cultivars at maturity. Our data indicate that deposition of some constituents of wax and cutin ceased during early fruit development.     

Effect of sample treatment on biomechanical properties of insect cuticle

Experimental limitations often prevent to perform biomechanical measurements on fresh arthropod cuticle samples. Hence, in many cases short- or long-term storage of samples is required. So far, it is not known whether any of the standard lab-techniques commonly used to fix or store insect cuticle samples in any way affects the biomechanical properties of the respective samples.In this paper we systematically address this question for the first time, with a focus on practical, easily accessible and common lab-methods including storage in water, ethanol, glutaraldehyde, freezing and desiccation. We performed a comprehensive and sensitive non-destructive Dynamic Mechanical Analysis (DMA) on locust hind leg tibiae using a three-point-bending setup. Our results show that from all tested treatments, freezing samples at −20 °C was the best option to maintain the original values for Young's modulus and damping properties of insect cuticle. In addition, our results indicate that the damping properties of locust hind legs might be mechanically optimized in respect to the jumping and kicking direction.     

Design of an insect cuticle associated with osmoregulation: the porous plates of chloride cells in a mayfly nymph

In mayfly nymphs of the genus Coloburiscoides, cell complexes with an osmoregulatory function (so-called chloride cells) are found in the integuments of the oral gills, the abdominal gills and gill filaments, the coxae and the thoracic sternites. The cuticle overlying each cell complex is a rigid circular plate which is known to be porous to colloidal lanthanum suspensions. The present study shows that the plate is composed only of the cuticulin and dense layers of the epicuticle. Both layers have substructures built of subunits on almost perfect hexagonal lattices. The lattice spacings are 53 and 9.5 nm for the dense layer and the cuticulin layer respectively. During moulting the apical plasma membrane of the chloride cell remains adpressed to the old porous plate. The new porous plate is formed from a new chloride cell which intrudes from the base of the integument. Throughout the moult small pores persist in the new and otherwise continuous cuticle to allow continuity of the cytoplasm of the apical and basal portions of the old chloride cell. It is thought that this phenomenon allows osmoregulatory function of the chloride cell complex to be maintained during the moult.     

Damage,repair and regeneration in insect cuticle: The story so far,and possibilities for the future

The exoskeleton of an insect can contain countless specializations across an individual, across developmental stages, and across the class Insecta. Hence, the exoskeleton's building material cuticle must perform a vast variety of functions. Cuticle displays a wide range of material properties which are determined by several known factors: the amount and orientation of the chitin fibres, the constituents and degree of cross-linking and hydration of the protein matrix, the relative amounts of exo- and endocuticle, and the shape of the structures themselves. In comparison to other natural materials such as wood and mammal bone, relatively few investigations into the mechanical properties of insect cuticle have been carried out. Of these, very few have focussed on the need for repair and its effectiveness at restoring mechanical stability to the cuticle. Insect body parts are often subject to prolonged repeated cyclic loads when running and flying, as well as more extreme “emergency” behaviours necessary for survival such as jumping, wedging (squeezing through small holes) and righting (when overturned). What effects have these actions on the cuticle itself? How close to the limits of failure does an insect push its body parts? Can an insect recover from minor or major damage to its exoskeleton “bones”? No current research has answered these questions conclusively.     

Superhydrophobic cuticle with a "pinning effect" in the larvae of the iris sawfly, Rhadinoceraea micans (Hymenoptera, Tenthredinidae)

The integument of some sawfly larvae can be easily damaged causing haemolymph bleeding (easy bleeding phenomenon). In the present study, we investigated intact cuticle, cuticle without wax coverage and cuticle replicas of sawfly larvae by using cryo-scanning electron microscopy and contact angle (CA) measurements. The easy bleeder Rhadinoceraea micans was compared to the non-easy bleeder Nematus pavidus (both Tenthredinidae), and the examination showed that the surface structures of R. micans were hierarchically arranged, whereas the cuticle surface of N. pavidus appeared much smoother. The intact cuticle proved to be superhydrophobic in R. micans (CAs: 156° dorsally, 120° ventrally), but not in N. pavidus (CAs: 67° dorsally, 47° ventrally). The wettability of R. micans increased significantly after the waxes had been peeled off. Replicas of the intact cuticle surface were hardly wettable in both species. It is suggested that wax crystals in combination with cuticle microsculptures are the most important features for rendering the integument of the easy bleeder species superhydrophobic. The wax-free tips of the cuticle microsculptures in R. micans are sites where haemolymph droplets can adhere. The integument surface of easy bleeders is assumed to be adapted to their particular defence strategy, simultaneously contributing to the maintenance and "reuse" of released haemolymph droplets on the superhydrophobic surface due to this "pinning effect".     

Effects of cuticle structure and crystalline wax coverage on the coloration in young and old males of Calopteryx splendens and Calopteryx virgo

Male secondary sexual characters, such as color patterns, are often investigated at the macroscale level. However, micro- and nanoscale levels of morphological investigations may reveal functional features responsible for a particular coloration, thus providing more information, e.g., about the condition dependence of male sexual characters. The aim of this paper was to investigate cuticle color and its structure in males of two congeneric damselfly species, Calopteryx splendens and Calopteryx virgo, and reveal possible color changes with age. According to spectrometer measurements, C. splendens males were bluer and had a greater saturation of blue in their abdomen than C. virgo males, which were, in turn, greener and had more green saturation. Although the two species differed in the number of structural layers and the spacing of the layers, it seems that intactness of the wax crystals covering the epicuticle was most often the morphological trait which was related to the color parameters measured from males’ cuticles. The effect of the crystalline wax coverage on cuticle color was also confirmed by removing the wax using chloroform: after the treatment, the hue was bluer, the cuticle had a greater brightness and greater blue saturation, but less green saturation. Age differences influencing the color and structure of the cuticle were also observed: older males had more blue and green saturation and had more intact wax coverage than did younger males. Although multilayer reflection should be responsible for the iridescent color of males, our results suggest that wax coverage plays an important role in the color tuning of the male cuticle. This may have a considerable signal function, indicating the males’ viability to competing males or to females.     

The eggshell of Drosophila melanogaster. VIII. Morphogenesis of the wax layer during oogenesis

Utilizing freeze-fracturing and conventional electron microscopy methods, we have studied the details of morphogenesis and construction of the wax layer envelope from Oregon R and mutants of Drosophila melanogaster egg' s during oogenesis. The wax layer is synthesized and secreted by the follicular cells in the 10b of lipid vesicles during static 10b. During secretion (stages 10b, 11 and 12) the lipid vcsicles are accumulated on the vitelline membrane surface and become flat. At the late stage of choriogenisis (stages 13, 14) the lipid vesicles are compressed tightly between the vitelline membrane and the other already constructed eggshell layers, so the wax layer becomes very thin and is hardly seen in crossfractured views.     

Ultrastructure and formation of the physogastric termite queen cuticle

The physogastric termite queen is the most striking example in insects of growth in size without cuticular moulting. This phenomenon has been studied with electron microscopy and histochemical tests in two species of higher termites, Cubitermes fungifaber and Macrotermes bellicosus. The abdominal hypertrophy (physogastry) is allowed by growth of the arthrodial membranes of the swarming imago. The growth is slow (over several years) but important: the cuticular dry weight is multiplied by 20 in C. fungifaber, by 100-150 in M. bellicosus. The termite queen cuticle arises from the transformation of the cuticle of the swarming imago or imaginal cuticle (unfolding and growing of the epicuticle, stretching of the endocuticle, resorption of the subcuticle) and from the secretion of a new endocuticle or royal endocuticle. The termite queen is the first example known in insects of epicuticular growth. In the physogastric queen, three cuticular types are observed: the rigid cuticle of the sclerites, the soft cuticle of the arthrodial membranes and the partially rigid cuticle of special structures, the neosclerites, which show both rigidity and growth. The fibrillar architecture varies according to the abdominal zones and the position within the cuticle. It appears to be determined by the forces arising from the musculature and the anisometric abdominal growth. The king does not become physogastric, although its cuticle is also modified.     

Antimicrobial activity of the Anseriform outer eggshell and cuticle

The avian eggshell is a complex, multifunctional biomineral composed of a calcium carbonate mineral phase and an organic phase of lipids and proteins. The outermost layer of the eggshell, the eggshell cuticle, is an organic layer of variable thickness composed of polysaccharides, hydroxyapatite crystals, lipids and glycoprotein. In addition to regulating gas exchanges, the eggshell cuticle may contain antimicrobial elements. In this study, we investigated the antimicrobial activity of eggshell cuticle and outer eggshell protein extracts from four Anseriform species: wood duck (Aix sponsa), hooded merganser (Lophodytes cucullatus), Canada goose (Branta canadensis) and mute swan (Cygnus olor). Cuticle and outer eggshell protein was extracted by urea or HCl treatment of eggs. C-type lysozyme, ovotransferrin and an ovocalyxin-32-like protein were detected in all extracts. Cuticle and outer eggshell protein extracts inhibited the growth of Staphylococcus aureus, Escherichia coli D31, Pseudomonas aeruginosa and Bacillus subtilis. The presence of active antimicrobial proteins within the avian cuticle and outer eggshell suggests a role in antimicrobial defense. Protein extracts from the cavity nesting hooded merganser were especially potent. The unique environmental pressures exerted on cavity-nesting species may have led to the evolution of potent antimicrobial defenses.     

Cadmium leads to stimulated expression of the lipid transfer protein genes in barley: implications for the involvement of lipid transfer proteins in wax assembly

In order to investigate the nature of genes expressed in leaf epidermal cells of higher plants, we have identified the nucleotide sequence of a cDNA designated ltp 7a2b encoding a novel nonspecific lipid transfer protein of barley (Hordeum vulgare L. cv. Gerbel). The cDNA of 755 basepairs contains an open reading frame of 366 nucleotides coding for a 12.3-kDa polypeptide. The first 29 amino acids constitute the putative signal peptide, characteristic for targeting to the secretory pathway. Analysis of mRNA levels by Northern blotting indicated that ltp 7a2b is preferentially expressed in the leaf epidermis. Levels of mRNA decreased during ageing of leaf tissue. Expression of ltp 7a2b was stimulated by a factor of 2–3 when the seedlings were grown in the presence of cadmium (10–1600 μM). Concomitantly, the primary leaves of Cd-exposed seedlings contained elevated levels of abscisic acid and a thicker wax layer of the cuticle. At 100 μM Cd in the hydroponic medium, the wax cover was increased by 50%. The increase in abscisic acid content, ltp 7a2b mRNA and wax coverage was either not seen, or seen much less, in Ni- and Zn-stressed seedlings. The data add circumstantial evidence to the recently proposed hypothesis that nonspecific lipid transfer proteins function in transfer of cutin and/or wax monomers from the site of synthesis in the cell to the cuticle. Received: 1 April 1996 / Accepted: 11 February 1997     

Ontogenetic and seasonal development of wax composition and cuticular transpiration of ivy (Hedera helix L.) sun and shade leaves

The ontogenetic and seasonal development of wax composition and cuticular transpiration of sun and shade leaves of ivy (Hedera helix L.) was analysed by investigating leaves varying in age between 4 and 202 d. It was discovered that the total amount of solvent-extractable wax was composed of two distinct fractions, separable by column chromatography: (i) a less polar or apolar monomeric wax fraction consisting of the typical linear, long-chain aliphatics usually described as cuticular wax components and (ii) a polar, oligomeric wax fraction consisting of primary alcohols and acids mostly esterified to C₁₂-, C₁₄- and C₁₆-ω-hydroxyfatty acids. The apolar wax fraction, which could be analysed directly by gas chromatography coupled with mass spectrometry (GC-MS), exhibited pronounced seasonal changes in composition. Wax amounts in the apolar fraction reached a maximum after about 30 d and gradually decreased again during the remaining period of the season investigated. In contrast, the polar wax fraction, which was analysable by GC-MS only after transesterification, rapidly increased early in the season, reaching a plateau after 40 d, and then remained constant during the rest of the season. Thus, total amounts of solvent-extractable cuticular waxes, which can be determined gravimetrically, will only be detected by GC-MS after fractionation and transesterification, a methodological approach rarely applied in the past in cuticular wax analysis. Additionally, investigation of the cutin polymer matrix after depolymerisation through transesterification, revealed that only those primary alcohols and acids forming an essential part of the apolar and the polar wax fractions were esterified during the investigated season and incorporated in increasing amounts into the cutin polymer matrix (matrix-bound wax fraction). Thus, it can be concluded that a complete analysis of cuticular wax of ivy and its seasonal development can only be achieved if all the relevant fractions (i) the less polar or apolar, (ii) the polar and (iii) the wax fraction bound to the cutin polymer matrix are investigated. Cuticular transpiration rapidly decreased within the first 30 d and essentially remained constant during the rest of the season. Thus, changes in cuticular water permeability were closely correlated with the most prominent changes in wax amounts and composition occurring during the first 30 d of ontogenetic leaf development. However, during the remainder of the year, up to 202 d, cuticular transport properties remained constant, although significant quantitative and qualitative changes in cuticular wax composition continued to occur. Thus, our study clearly demonstrated that there will be no simple relationship between chemical composition of cuticular waxes and transport properties of isolated ivy leaf cuticles. Received: 2 March 1998 / Accepted: 26 June 1998     

The ultrastructural organization of the visual system of the wax moth,Galleria mellonella: The retina

Summary The ultrastructural organization of ommatidial components of the retina of the moth, Galleria mellonella are described from electron microscopic observations. Each ommatidium is composed of 12 common retinula cells and one basal eccentric cell. The retinula cells are connected together by a desmosomal strip along their length. The rhabdom occupies the basal thirty percent of the ommatidium and can be divided into nine segments of parallel microvilli. Several cells may contribute to an individual rhabdomere. The rhabdomeres are arranged in a cross with single cell rhabdomeres lying between the arms of the cross. Thin sections of ommatidium absorb polarized light differentially. The total amount of plane polarized light absorbed varies with angle of rotation for an entire ommatidium but there are also differences between the amount of absorption of adjacent rhabdomeric segments. Galleria appears to be the only lepidopteran in which the possibility of the polarized light reception has been reported.     

Involvement of Yellow-y in the cuticle pigmentation of the larvae,pupae and adults in Henosepilachna vigintioctopunctata

Yellow-y (Y-y) contributes to the accumulation of melanins in insect cuticle. However, the underlining mechanism requires further investigation. Two classical hypotheses have been proposed: Y-y acts as a dopachrome conversion enzyme (DCE) to accelerate biosynthesis of melanins; alternatively, Y-y serves as a cuticular anchor for pigments. Henosepilachna vigintioctopunctata is a serious defoliator attacking Solanaceae and Cucurbitaceae plants. The beetle shows a species-specific pigmentation pattern: stage-dependent dark patches are distributed on pale-yellow background. Here we noted that RNA interference (RNAi)-aided knockdown of Hvyellow-y at the newly-ecdysed second- and third-instar larval, and 1-day-old prepupal stages changed coloration in both dark patches and pale-yellow background. Black pigmentation was lightened in the Hvy-y hypomorphs, including various body portions such as larval heads, antennae, mouthparts, scoli, strumae, legs and exuviae, pupal and adult thoraces and abdomens, and adult elytra and hindwings. Moreover, the coloration background was yellowed in the RNAi beetles. Specifically, more yellow pigments were observed to deposit around the black dorsal markings in the hypomorphic pupal metathorax. Furthermore, the boundaries between black patches and yellow background were distinct in the resultant ladybirds. Similarly, the margins around bristle follicles and droplet spots were not fuzzy within the RNAi pupal black patches. In summary, even though Y-y facilitates the pigmentation in H. vigintioctopunctata exocuticle, our data did not support the pigment anchor hypothesis.     

Cuticular wax deposition in growing barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) leaves commences in relation to the point of emergence of epidermal cells from the sheaths of older leaves

In grasses, leaf cells divide and expand within the sheaths of older leaves, where the micro-environment differs from the open atmosphere. By the time epidermal cells are displaced into the atmosphere, they must have a functional cuticle to minimize uncontrolled water loss. In the present study, gas chromatography and scanning electron microscopy were used to follow cuticular wax deposition along the growing leaf three of barley (Hordeum vulgare L.). 1-Hexacosanol (C₂₆ alcohol) comprised more than 75% of extractable cuticular wax and was used as a marker for wax deposition. There was no detectable wax along the first 20 mm from the point of leaf insertion. Deposition started within the distal portion of the elongation zone (23–45 mm) and continued beyond the point of leaf emergence from the sheath of leaf two. The region where wax deposition commenced shifted towards more proximal (basal) positions when the point of leaf emergence was lowered by stripping back part of the sheath. When relative humidity in the shoot environment was elevated from 70% (standard growth conditions) to 92–96% for up to 4 days prior to analysis, wax deposition did not change significantly. The results show that cuticular waxes are deposited along the growing grass leaf independent of cell age or developmental stage. Instead, the reference point for wax deposition appears to be the point of emergence of cells into the atmosphere. The possibility of changes in relative humidity between enclosed and emerged leaf regions triggering wax deposition is discussed.