Phenolic constituents of tomato fruit cuticles

Chalconaringenin, naringenin, naringenin-7-glucoside, and m- and p-coumaric acids have been identified in the fruit cuticles of three tomato cultivars. The phenolic content of the cuticles increased substantially during fruit development, those from immature green and mature ripe fruits of cv Ailsa Craig yielding respectively 2.8 and 61 μg/cm² (representing 1.4 and 6% of the total membrane wt). Coumaric acids, present only in the ‘cutin-bound’ phenolics, increased from 2 to 24 μg/cm² during fruit development. Flavonoids, synthesized mainly during the climacteric, occurred free in the epicuticular (0.3–7.2 μg/cm²) and cuticular (0.7–5.7 μg/cm²) phenolics but the major part of this class of constituents in ripe fruit cuticles was also ‘bound’ to the cutin matrix (30–43 μg/cm²). The composition of the flavonoid fraction was controlled by the spectral quality of incident radiation, red light favouring the formation of chalconaringenin.     

Water permeability of plant cuticles: permeance,diffusion and partition coefficients

Summary Using isolated cuticular membranes from ten woody and herbaceous plant species, permeance and diffusion coefficients for water were measured, and partition coefficients were calculated. The cuticular membranes of fruit had much higher permeance and diffusion coefficients than leaf cuticular membranes from either trees or herbs. Both diffusion and partition coefficients increased with increasing membrane thickness. Thin cuticles, therefore, tend to be better and more efficient water barriers than thick cuticles. We compared the diffusion coefficients and the water content of cuticles as calculated from transport measurements with those obtained from water vapor sorption. There is good to fair agreement for cuticular membranes with a low water content, but large discrepancies appear for polymer matrix membranes with high permeance. This is probably due to the fact that diffusion coefficients obtained from transport measurements on membranes with high permeance and water content are underestimated. Water permeabilities of polyethylene and polypropylene membranes are similar to those of leaf cuticular membranes. However, leaf cuticles have much lower diffusion coefficients and a much greater water content than these synthetic polymers. This suggests that cuticles are primarily mobility barriers as far as water transport is concerned.     

Cuticular proteins: The neglected component

This paper emphasizes the importance of the protein component of cuticles. Correlation of electrophoretic charge distribution of individual cuticular proteins and physical properties of the cuticles from which they were extracted, as well as interpopulation and interspecies conservation of electrophoretic patterns, are used to argue that individual proteins play precise roles in the cuticle. Glycosylation of cuticular proteins is described, but no function for these modifications is yet known. Analogy is drawn to analyses of chorion proteins and the case is made that analysis of amino acid sequence data is likely to provide insights into how cuticular proteins and chitin interact to construct the diverse types of cuticles.     

AgCl precipitates in isolated cuticular membranes reduce rates of cuticular transpiration

Counter diffusion of chloride, applied as NaCl at the inner side of isolated cuticles, and silver, applied as AgNO₃ at the outer side, lead to the formation of insoluble AgCl precipitates in isolated cuticles. AgCl precipitates could be visualized by light and scanning electron microscopy. The presence of AgCl precipitates in isolated cuticles was verified by energy dispersive X-ray analysis. It is argued that insoluble AgCl precipitates formed in polar pores of cuticles and as a consequence, cuticular transpiration of 13 out of 15 investigated species was significantly reduced up to three-fold. Water as a small and uncharged but polar molecule penetrates cuticles via two parallel paths: a lipophilic path, formed by lipophilic cutin and wax domains, and a aqueous pathe, formed by polar pores. Thus, permeances P (m s⁻¹) of water, which is composed of the two quantities P _Lipid and P _Pore, decreased, since water transport across polar pores was affected by AgCl precipitates. Cuticles with initially high rates of cuticular transpiration were generally more sensitive towards AgCl precipitates compared to cuticles with initially low rates of transpiration. Results presented here, significantly improves the current model of the structure of the cuticular transpiration barrier, since the pronounced heterogeneity of the cuticular transport barrier, composed of lipophilic as well as polar paths of diffusion, has to be taken into account in future.     

Development of plant cuticles: occurrence and role of non-ester bonds in cutin of Clivia miniata Reg. leaves

The effect of BF₃-methanol treatment on the mass and fine structure of isolated Clivia leaf cuticles at different stages of development has been investigated. BF₃-methanol cleaves ester linkages in cutin; however, the cuticles are not completely depolymerized. With increasing age, the residue left after BF₃-methanol treatment increases in mass. In very young cuticles, 10% of the total cutin resisted BF₃-methanol and the fraction of nonester cutin increased up to 62% in mature leaves. Transmission electron microscopy shows that fine structure of the cuticle proper is severely distorted but not destroyed. The internal cuticular layer, which exhibits a heavy contrast when fixed with KMnO₄, is completely depolymerized, while the external cuticular layer is hardly affected. The results are discussed in relation to cuticle development and to the function of cuticles as transpiration resistances.Abbreviation CP cuticle proper - ECL external cuticular layer - E cutin ester bonded cutin - ICL internal cuticular layer - MX-membrane polymer matrix membrane - NE-cutin non-ester bonded cutin - TEM transmission electron microscopy     

Post-translational modifications of the cuticular proteins of Hyalophora cecropia from different anatomical regions and metamorphic stages

Post-translational modifications are a conspicuous feature of the proteins of vertebrate extracellular matrices such as cartilage. Yet this feature remains virtually unexplored with insect cuticle, a situation this paper begins to remedy. Cuticular proteins were extracted from cuticles of Hyalophora cecropia and separated on isoelectrofocusing and 2D gels. Periodic acid-Schiff reagent stained several proteins from flexible cuticles and a few proteins from rigid cuticles, indicating that some proteins were glycosylated. Elucidation of the specific nature of this glycosylation came from probing electrophoretically separated cuticular proteins blotted onto nitrocellulose with biotinylated lectins. Most major cuticular proteins did not react; minor cuticular proteins and molecules which do not stain with Coomassie blue were found to bind lectins specific for mannose and N-acetylgalactosamine. Limited binding was also detected with lectins specific for N-acetylglucosamine, galactose and fucose. No sialic acid was detected using either lectins or neuraminidase digestion. The amount of glycosylation was greatest in proteins extracted from flexible cuticles. Although several proteins stained with Alcian blue indicating presence of sulfation, ³⁵S which had been incorporated at low levels in cuticular proteins corresponded to [³⁵S]methionine. No indication of the presence of mammalian-type glycosaminoglycans in insect cuticles was obtained after treatment with chondroitinase or nitrous acid. The functional significance of the modifications detected remains unknown. No evidence for phosphorylated proteins or lipoproteins was found.     

Analysis of the cuticular proteins of Hyalophora cecropia with polyclonal antibodies

The cuticular proteins from different anatomical regions and metamorphic stages of Hyalophora cecropia were analyzed with polyclonal antibodies raised against cuticular protein extracts from each stage. Western blots of 2D gels coupled with detection of antibody-antigen binding with avidin-biotinylated-horseradish peroxidase complexes (ABC method) proved to be extremely sensitive. Reactions of polyclonal antisera with blots of extracts of different cuticular regions revealed the following: (1) glycosylated cuticular proteins were highly antigenic; (2) there was less cross-reaction between rigid and flexible cuticles from the same metamorphic stage than among cuticles with similar mechanical properties from different stages; (3) proteins with identical molecular weights and isoelectric points were antigenically indistinguishable.     

Flavonoid biosynthesis in tomato fruit cuticles after in vivo incorporation of 3 H-phenylalanine precursor

Radiolabelling of epicuticular waxes and cutin of isolated tomato fruit cuticles were determined after fruit surface application of 3 H-phenylalanine precursor. During fruit ripening, the precursor is incorporated in different phenolic components: the flavanone naringenin was found to be the major compound in the epicuticular waxes, while the amount of the labelled flavonoid in the cutin matrix was progressively increased throughout fruit ripening. Confocal microscopy, together with experimental estimation of the mobility (diffusion coefficient, D ) and affinity (partition coefficient, K ) of the flavonoids naringenin and chalconaringenin for the different cuticular components, indicate that these compounds are extruded to the outer surface of tomato fruits, forming molecular clusters.     

Water transport in plant cuticles: an update

The scale, mechanism, and physiological importance of cuticular transpiration were last reviewed in this journal 5 and 10 years ago. Progress in our basic understanding of the underlying processes and their physiological and structural determinants has remained frustratingly slow ever since. There have been major advances in the quantification of cuticular water permeability of stomata-bearing leaf and fruit surfaces and its dependence on leaf temperature in astomatous surfaces, as well as in our understanding of the respective roles of epicuticular and intracuticular waxes and molecular-scale aqueous pores in its physical control. However, understanding the properties that determine the thousand-fold differences between permeabilities of different cuticles remains a huge challenge. Molecular biology offers unique opportunities to elucidate the relationships between cuticular permeability and structure and chemical composition of cuticles, provided care is taken to quantify the effects of genetic manipulation on cuticular permeability by reliable experimental approaches.     

Water permeability of plant cuticles: The effect of temperature on diffusion of water

The effect of temperature on water permeability of plant cuticles (astomatous Citrus leaf cuticles) has been investigated. The Arrhenius plot (logarithm of the permeability coefficient vs. 1/temperature) has two linear portions that intersect at 44° C. Evidence is presented to show that this intersection represents the solid/liquid phase transition of cuticular lipids. As the Arrhenius plot has only one phase transition in the temperature range of 5 to 80° C, it appears that all soluble cuticular lipids in the cuticle are present as a homogeneous mixture rather than as individual layers differing in composition. This view is supported by electron spin resonance evidence showing homogenous distribution of spin label fatty acids. The original distribution of soluble cuticular lipids is irreversibly altered by heating cuticular membranes above the transition temperature. This is accompanied by an irreversible increase in water peremeability, demonstrating the importance of the structure of cuticular lipids with regard to cuticular permeability.Abbreviations CM cuticular membranes - MX polymer matrix - SCL soluble cuticular lipids - MES morpholinoethane sulphonic acid - J flux - ESR electron spin resonance - THO tritiated water     

Cuticular Pores and Transcuticular Canals in Diverse Fruit Varieties

Dewaxed thin-sectioned and dewaxed isolated mature fruit cuticlesrevealed the unequivocal presence in situ of visibly discrete,ubiquitous, cuticular pores or orifices concomitant with anticlinally-orientedtranscuticular canals in 51 varieties of fruit among 20 plantfamilies. More than 66 per cent of the fruit cuticles have poresand/or canals. No correlation exists between either fruit sizeor pore size and cuticle thickness. Dewaxed cuticles rangedfrom 1.25–22.5 µm in thickness. Canal lengths aredirectly related to cuticle thickness. Cuticular occlusionsof the epidermal cells were found in 76 per cent of the fruitsexamined. Evidence is provided by light microscopy photomicrographs. Fruit cuticles, cuticle morphology, cuticular pores, transcuticular canals     

大叶藤黄叶片角质层的酶分离技术

角质层运输特征的研究在生态学、环境和农用化学等方面都具有非常重要的意义 ,完好地分离角质层是进行这方面研究的前提。本文以生长在西双版纳热带雨林中的大叶藤黄为材料 ,探讨了角质层的酶分离技术。结果表明 ,30℃时用 2 % (w/v) ,pH 3 0的果胶酶和纤维素酶溶液浸泡大叶藤黄叶圆片 9h ,叶圆片边缘角质层分离 ,浸泡 5d可分离到完整的角质层 ,角质层在硼酸盐缓冲液 (0 0 1mol·L-1,pH 9 0 )中充分浸泡数日去除石炭酸 ,空气中干燥 ,室温下于聚乙烯的盒子保存备用     

Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry

Identification of authenticated cuticular proteins has been based on isolation and sequencing of individual proteins extracted from cleaned cuticles. These data facilitated classification of sequences from conceptual translation of cDNA or genomic sequences. The question arises whether such putative cuticular proteins actually are incorporated into the cuticle. This paper describes the profiling of cuticular proteins from Anopheles gambiae starting with cuticle cleaned by the insect itself in the course of molting. Proteins extracted from cast larval head capsules and cast pupal cuticles were fractionated by 1D SDS gel electrophoresis. Large gel slices were reduced, carbamidomethylated and digested with trypsin. The pellet remaining after SDS extraction was also treated with trypsin. The resulting peptides were separated on a C18 column and then analyzed by tandem mass spectrometry. Two-hundred-ninety-five peptides from putative cuticular proteins were identified; these corresponded to a minimum of 69 and a maximum of 119 different proteins. Each is reported as an authentic Anopheles cuticular protein for the first time. In addition to members of two known cuticular protein families, members of additional families likely to be structural components of the cuticle were identified. Furthermore, other peptides were identified that can be attributed to molting fluid, muscle and sclerotizing agents.     

Consideration of the importance of hydrophobic interactions in stabilizing insect cuticle

Protein fractions of insect cuticles with different mechanical properties have related values of polarity and hydrophobicity. Hydrophobicity is important for the self-assembly of cuticle which is produced prior to the moult and in plasticization of cuticle. The cuticles of soft-bodied fly larvae are quite distinct from those of exopterygotes (e.g. locusts) and this can be related to the chemistry and mode of tanning. The properties of cuticular proteins are compared: the proteins of the pliant cuticles most closely resemble globulins, and the proteins in stiff cuticles are more like fibrous and hydrophobic structural proteins. Changes in the environment of the proteins may alter their shape and hence the mechanical properties of the cuticle.     

Characterization of aqueous pores in plant cuticles and permeation of ionic solutes

Plant cuticles are lipid membranes with separate diffusion paths for lipophilic non-electrolytes and hydrated ionic compounds. Ions are lipid insoluble and require an aqueous pathway across cuticles. Based on experimental data, the aqueous pathway in cuticles has been characterized. Aqueous pores arise by hydration of permanent dipoles and ionic functional groups. They can be localized using ionic fluorescent dyes, silver nitrate, and mercuric chloride. Aqueous pores preferentially occur in cuticular ledges, at the base of trichomes, and in cuticles over anticlinal walls. Average pore radii ranged from 0.45 to 1.18 nm. Penetration of ions was a first order process as the fraction of the salt remaining on the cuticle surface decreased exponentially with time. Permeability of cuticles to ions depended on humidity and was highest at 100% humidity. Wetting agents increased rate constants by factors of up to 12, which indicates that the pore openings are surrounded by waxes. The pores in cuticular ledges of Helxine soleirolii allowed passage of berberine sulphate, which has a molecular weight of 769 g mol(-1). Increasing the molecular weight of solutes from 100 to 500 g mol(-1) decreased the rate constants of penetration by factors of 7 (Vicia faba) and 13 (Populus canescens), respectively. Half-times of penetration of inorganic salts and organic ions across Populus cuticles and Vicia leaf surfaces varied between 1 and 12 h. This shows that penetration of ionic compounds can be fairly rapid, and ions with molecular weights of up to 800 g mol(-1) can penetrate cuticles that possess aqueous pores.     

The Prevalence of Pores and Canals in Leaf Cuticular Membranes

Ubiquitous, visibly discrete, natural cuticular pores and transcuticularcanals were found in the dewaxed leaf (and one herbaceous stem)cuticular membranes of 27 out of 32 taxa among 14 families.Clear evidence for their existence is provided by light photomicrographs.Both adaxial and abaxial leaf surfaces were investigated usingthin transections and chemically isolated cuticular membranes,in conjunction with ordinary staining techniques and light microscopymethods. No correlations were found between cuticle thicknessesand either the frequency of pore or the pore and canal diameters. Leaf cuticles, cuticle morphology, cuticular pores, transcuticular canals, cuticular flanges     

Protecting against water loss: analysis of the barrier properties of plant cuticles

The cuticle is the major barrier against uncontrolled water loss from leaves, fruits and other primary parts of higher plants. More than 100 mean values for water permeabilities determined with isolated leaf and fruit cuticles from 61 plant species are compiled and discussed in relation to plant organ, natural habitat and morphology. The maximum barrier properties of plant cuticles exceed that of synthetic polymeric films of equal thickness. Cuticular water permeability is not correlated to the thickness of the cuticle or to wax coverage. Relationships between cuticular permeability, wax composition and physical properties of the cuticle are evaluated. Cuticular permeability to water increases on the average by a factor of 2 when leaf surface temperature is raised from 15 degrees C to 35 degrees C. Organic compounds of anthropogenic and biogenic origin may enhance cuticular permeability. The pathway taken by water across the cuticular transport barrier is reviewed. The conclusion from this discussion is that the bulk of water diffuses as single molecules across a lipophilic barrier while a minor fraction travels along polar pores. Open questions concerning the mechanistic understanding of the plant cuticular transport barrier and the role the plant cuticle plays in ensuring the survival and reproductive success of an individual plant are indicated.     

Partial amino acid sequences of cuticular proteins from Hyalophora cecropia

The amino-terminal amino acid sequences for seven cuticular proteins from Hyalophora cecropia are reported. Proteins were purified by blotting two dimensional acrylamide gels onto acid-etched glass fiber filters, and the proteins were sequenced without further elution. The sequences of the serine-rich proteins from rigid cuticles revealed a new family of cuticular proteins, with features reminiscent of the amino-termini of certain vertebrate neurofilament proteins, members of the intermediate filament protein family which includes keratins. The proteins from flexible cuticles showed sequence similarity to proteins previously sequenced for Drosophila, Manduca and Sarcophaga. Proteins with identical electrophoretic mobility from two different metamorphic stages or from two anatomical regions within a single stage had identical amino-terminal sequences.     

New insights into the properties of pubescent surfaces: peach fruit as a model

The surface of peach (Prunus persica 'Calrico') is covered by a dense indumentum, which may serve various protective purposes. With the aim of relating structure to function, the chemical composition, morphology, and hydrophobicity of the peach skin was assessed as a model for a pubescent plant surface. Distinct physicochemical features were observed for trichomes versus isolated cuticles. Peach cuticles were composed of 53% cutan, 27% waxes, 23% cutin, and 1% hydroxycinnamic acid derivatives (mainly ferulic and p-coumaric acids). Trichomes were covered by a thin cuticular layer containing 15% waxes and 19% cutin and were filled by polysaccharide material (63%) containing hydroxycinnamic acid derivatives and flavonoids. The surface free energy, polarity, and work of adhesion of intact and shaved peach surfaces were calculated from contact angle measurements of water, glycerol, and diiodomethane. The removal of the trichomes from the surface increased polarity from 3.8% (intact surface) to 23.6% and decreased the total surface free energy chiefly due to a decrease on its nonpolar component. The extraction of waxes and the removal of trichomes led to higher fruit dehydration rates. However, trichomes were found to have a higher water sorption capacity as compared with isolated cuticles. The results show that the peach surface is composed of two different materials that establish a polarity gradient: the trichome network, which has a higher surface free energy and a higher dispersive component, and the cuticle underneath, which has a lower surface free energy and higher surface polarity. The significance of the data concerning water-plant surface interactions is discussed within a physiological context.     

Expression and hormonal control of a new larval cuticular multigene family at the onset of metamorphosis of the tobacco hornworm

The pattern of cuticular protein synthesis by the epidermis of the tobacco hornworm larva changes during the final day of feeding, leading to an alteration in cuticular structure and a stiffening of the cuticle. We have isolated a small multigene family which codes for at least three of the new cuticular proteins made at this time. The five genes which were isolated from this family map to two different genomic regions. Sequencing shows that one of the genes is 1.9 kb and consists of three exons coding for a 12.2-kDa acidic (pI = 5.26) protein that is predominantly hydrophilic. The deduced amino acid sequence shows regions of similarity to proteins from flexible lepidopteran cuticles and from Drosophila larval and pupal cuticles, but not to proteins found in highly sclerotized cuticles. This gene family is first expressed late on the penultimate day (Day 2) of feeding in the final larval instar and ceases expression 2 days later when metamorphosis begins. In situ hybridization shows that this gene family is expressed in all the epidermal cells of Day 3 larvae except the bristle cells and those at the muscle attachment site. Expression can be induced in Day 1 epidermis by exposure to 50 ng/ml 20-hydroxyecdysone in vitro, but only if juvenile hormone is absent. Its developmental expression, tissue specificity, and hormonal regulation strongly suggest that this multigene family is involved in the structural changes that occur in the larval cuticle just prior to the onset of metamorphosis.