Apple Fruit Cuticles and the Occurrence of Pores and Transcuticular Canals

Developmental studies were made on the dewaxed thin-sectionedapple fruit cuticles of 10 Malus sylvestris Mill, cultivarsfrom 4 weeks before anthesis through fruit maturation and harvest.Cuticular development appears to correlate well with the generalgrowth of the fruit. However, no correlation exists betweenfruit size and cuticle thickness. Cuticular pores were evidentas early as 1 week before anthesis and transcuticular canalsbecame evident by 1 week following anthesis. Dewaxed thin-sectioned,as well as isolated, mature fruit cuticles of 16 cultivars andfour crab-apples consistently revealed the distinct presenceof ubiquitous pores and canals. Evidence is provided by lightmicroscopy and scanning electron microscopy (SEM). Measurementswere taken of cuticle thicknesses during development and ofcuticular pore dimensions, and calculations were made of poreand canal numbers. Fruit size alone is not directly indicativeof total pore numbers per surface area. Canal lengths are directlyrelated to the developmental thickness of the cuticle. No correlationwas found between the thickness of the mature cuticle and eitherthe number of pores present or the pore diameters. Malus sylvestris Mill, apple fruit, morphology, cuticle, cuticular flanges, cuticular pores, transcuticular canals, ultrastructure     

The Morphology and Permeability of Isolated Cuticular Membranes of Hoya carnosa R. Br. (Asclepiadaceae)

This investigation is in part an extension of previous leafcuticle observations made on 52 other taxa among 34 families.Dewaxed, chemically isolated, adaxial and abaxial cuticularmembranes and transverse leaf sections of the wax-flower plant(Hoya carnosa R. Br.) were examined using ordinary stainingtechniques and light-microscopy methods. Evidence is presentedfor the existence of ubiquitous, discrete, naturally occurringcuticular pores, concomitant with anticlinally oriented trans-cuticularcanals, distributed randomly throughout the cuticular matrix.The surface of the adaxial cuticular membrane contains approx.6540 unclustered pores per mm², the abaxial approx. 4680 poresper mm². Pore and canal diameters range between 0.5 and 0.75µm. The canals are often arcuate and their lengths aredirectly related to cuticle thickness. No correlations werefound between cuticle thickness and either pore numbers or poreand canal diameters. Based upon experiments with various pHindicators, solutions, and stains, the dewaxed, dry cuticularmembrane of H. carnosa appears to be both distinctly hydrophilicand selectively permeable through a myriad of microscopicallyvisible pores and canals permeating its matrix. A de novo interpretationof gross cuticle morphology based solely upon light microscopyobservations is presented by semi-diagrammatic illustrations. Hoya carnosa R. Br., wax-flower (wax-plant), cuticular membranes, cuticular pores, transcuticular canals, permeability     

The Prevalence of Pores and Canals in Leaf Cuticular Membranes. 2. Supplemental Studies

Evidence is presented for the existence of discrete, naturalcuticular pores concomitant with anticlinally-oriented transcuticularcanals found in the mature leaf surfaces of 26 out of 37 taxaamong 19 families. This investigation is an extension of earlierobservations made on 32 other taxa among 14 families. Dewaxed,chemically isolated, adaxial and abaxial cuticular membranesin conjunction with transverse leaf sections were examined usingordinary staining techniques. The ubiquitous pores occur randomlywith no evidence of clustering. Pore and canal diameters averageapprox. 1 µm. Canal lengths are directly related to cuticlethickness. No correlations were found between cuticle thicknessesand either pore frequencies or pore and canal diameters. Evidenceis provided by light microscopy photomicrographs. Leaf cuticles, cuticular membranes, cuticular pores, transcuticular canals, cuticular flanges     

The Multiple Epidermis-Cuticle Complex of Medlar Fruit Mespilus germanica L. (Rosaceae)

In order to determine the authenticity of the multiple epidermallayers in the pomaceous fruit of Medlar or Mespil (Mespilusgermanica L.), developmental studies were made from 2 weeksbefore anthesis through harvest and storage. Various histochemicaltechniques were used for the determination of epidermal cellwall structures and cell contents, and for the isolation andstudy of the cuticular membranes. The multiseriate epidermisis derived from successive tangential divisions of the initiallyuniseriate epidermis commencing about 3 weeks post-anthesis.The divisions occur simultaneously around the fruit circumferenceand result in the formation of a normally four- to five-layeredepidermis at fruit maturity. As each epidermal layer is derivedit concomitantly develops a distinct, flanged cuticle that remainspersistent until sloughing of the layer occurs. Evidence providedby light and scanning electron microscopy indicates the presenceof discrete ubiquitous pores and non-anastomosing, anticlinallyoriented canals in the cuticular membranes. Measurements weremade of individual cuticular membrane thicknesses during development,and of cuticular pore dimensions in the mature fruit; cuticularpore numbers were calculated. A brief study was made of therelationship between lenticel development and development ofthe multiseriate epidermis. Mespilus germanica L., Medlar (Mespil) fruit, multiseriate epidermis, cuticular membrane, cuticular pores, transcuticular canals, lenticels     

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.     

Ultrastructure and Radiolabelling of Leaf Cuticles from Ivy (Hedera helixL.) Plantsin vitroand duringex vitroAcclimatization

Cuticle ultrastructure and radiolabelling of isolated cuticlesafter incorporation of [¹⁴C] acetate in foliar discs were investigatedwith ivy plants grownin vitrothenex vitro. Results show an increasein thickness, mass and wax content, between young and expandedleaves, for bothin vitroandex vitrocuticles. The cuticle ofinvitrounexpanded leaves was very thin and only constituted alamellate zone. The ultrastructure ofin vitroyoung and expandedleaf cuticles showed characteristics similar toin situcuticles.The thickness of the lamellate zone remained fairly constantand represented 33% of the cuticle thickness in young leaves,but only 11.4% in expanded leaves. The number of lamellar unitsdecreased from 14 to nine between these two growth stages. Themain difference between young leaves developedin vitroorex vitrowasa thinner lamellate zone forex vitrocuticles. However, theselatter cuticles had an intermediary zone between the lamellateand reticulate zones. The cuticle thickness of expanded leaveswas greater forin vitrocuticles suggesting a temporary decreasein cuticle biosynthesis after transfer of the plant fromin vitrotoexvitro.Results from cuticle radiolabelling show higher radioactivityincorporation in cuticles isolated from leaves developedex vitrocomparedtoin vitro. This radiolabelling was particularly marked forexvitroyoung leaf cuticles and depended on the duration of theexvitrogrowth period revealing a progressive activation of cuticlebiosynthesis in response to new environmental conditions. Hedera helix; ivy leaf cuticle; in vitroplants; electron microscopy; radiolabelling; isolated cuticles     

A simple photometric device analysing cuticular transport physiology: surfactant effect on permeability of isolated cuticular membranes of Prunus laurocerasus L.

4-Nitrophenol permeabilities of astomatous cuticular membranesisolated from the upper surface of Prunus laurocerasus L. leaveswere measured applying a newly developed photometric device.Isolated cuticles were mounted between donor and receiver compartmentsof a stainless steel transport chamber. 4-Nitrophenol was appliedas non-dissociated species in citric buffer at pH 3.0 in thedonor compartment and sampled as dissociated species in thereceiver compartment in borate buffer at pH 9.0. Permeances,calculated from steady-state rates of 4-nitrophenol permeation,ranged from 1.73 10^–10 m s^–1 up to 38.410^–10ms^–1. They were in the same order of magnitude comparedto published permeances obtained with a different method usingradiolabelled 4-nitrophenol and isolated cuticles of Citrusaurantium L. In the presence of the surfactant Brij 30, whichis a polydisperse alcohol ethoxylate, cuticular permeabilitiesincreased on average by a factor of 37. Cuticles, initiallyhaving the lowest permeabilities, exhibited the highest increaseof their permeabilities due to the surfactant and vice versa.This increase ofcuticular permeabilities in the presence ofa surfactant is interpreted as a plasticizing effect of thesurfactant molecules on the cuticular wax forming the cuticulartransport barrier. Furthermore, surfactant-induced increasesof cuticular permeabilities were reversible to a large extent.Permeabilities decreased again after the removal of Brij 30reaching final values about 6-times higher compared to the initialpermeabilities. This demonstrates that the surfactant and thepermeating molecule must be present simultaneously in the cuticlein order to enhance cuticular permeation. Possible applicationsof this simple photometric device analysing further aspectsof cuticular transport physiology are finally suggested. Key words: Cuticular transport, leaf surface, permeability, plant cuticle, surfactant     

Cuticular water permeability and its physiological significance

Cuticles act as solution-diffusion membranes for water transport.Diffusion in pores does not contribute to cuticular transpiration.An extensive literature survey of cuticular permeances (P) andminimum leaf conductances (g_min) to water is presented. Thetwo variables cannot be distinguished with most experimentaltechniques. Results from different experiments are in good agreementwith each other for some species, for example, Fagus sytvaticaL., but not for others, such as Picea abies (L.) Karst. In adata set of 313 values of P or g_min from 200 species, distributionsof results obtained with different techniques were found todiffer significantly. Likely reasons include water loss fromincompletely closed or incompletely sealed stomata, and thedependence of P on moisture content of the cuticle and on storagetime of isolated cuticles. Contrasting evidence for an interactionbetween cuticular transpiration and stomatal sensitivity toair humidity is presented. The occurrence of unusually highg_min in trees growing at the alpine treeline and its physiologicalsignificance are discussed. It is shown that g_min is of littlevalue as a predictor for drought resistance of crops, with thepossible exception of Sorghum bicolor L. Moench. Possible wateruptake from fog or dew across cuticles is considered briefly. Key words: Epidermal conductance, VPD-response, water absorption, waxes, winter desiccation     

La formation des canaux cuticulaires chez l'adulte de Tenebrio molitor L. étude ultrastructurale et remarques histochimiques

The sclerotized cuticle of adult Tenebrio shows (1) an exocuticle composed of rotating lamellate layers and of columns of cuticular material, the fibres of which run perpendicularly through the lamellae, (2) an endocuticle composed of layers with preferred orientation. In the exocuticle, the pore canals are numerous and run along the columns; they do not rotate with the lamellate layers. They show several filaments some of which leave the canals and form a dense intracuticular network. In the last layers of exocuticle, the pericolumnar canals fuse and form large endocuticular canals which rotate in phase with the cuticular fibres. The formation of columns and canals is in relation with cellular expansions which penetrate into the cuticle during cuticle deposition. Exocuticular columns seem characteristic of highly sclerotized cuticles and the intracuticular filaments may have a role in the transport of sclerotisation precursors.     

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     

Polar paths of diffusion across plant cuticles: new evidence for an old hypothesis

BACKGROUND: The plant cuticle is an extracellular lipophilic biopolymer covering leaf and fruit surfaces. Its main function is the protection of land-living plants from uncontrolled water loss. In the past, the permeability of the cuticle to water and to non-ionic lipophilic molecules (pesticides, herbicides and other xenobiotics) was studied intensively, whereas cuticular penetration of polar ionic compounds was rarely investigated. RECENT PROGRESS: Recent work measuring cuticular penetration of inorganic and organic ions is presented; the effects of molecular size of ions, temperature, wax extraction, humidity and plasticizers strongly support the conclusion that ions penetrate cuticles via water-filled pores. The cuticle covering stomata and trichomes forms the preferential site of ion penetration. This indicates that cuticles possess a pronounced lateral heterogeneity: the largest fraction of the cuticle surface is covered by the lipophilic domains of cutin and wax, but to a certain extent polar domains are also present in the cuticle, which form preferential sites of penetration for polar compounds. THE FUTURE: The chemical nature of these polar domains awaits detailed characterization, which will be of major importance in agriculture and green biotechnology, since polar paths of diffusion represent the most important transport routes for foliar-applied nutrients. Furthermore, many compounds acting as inducers of gene expression in transgenic plants are ionic and need to penetrate the cuticle via polar paths in order to be active.     

BINDING SITES FOR INORGANIC IONS AND UREA ON ISOLATED CUTICULAR MEMBRANE SURFACES

Binding sites for calcium and chloride ions, and urea on cuticular membrane surfaces were identified by microautoradiography. There was no localization of binding on either the smooth outer or irregular inner surfaces of the astomatous tomato fruit cuticle, and urea was bound to a lesser degree than inorganic ions. Calcium and chloride binding sites on surfaces of green onion leaf cuticles were closely associated with stomatal pores and above the periclinal cell walls. Binding of calcium was more pronounced than of chloride. These results are discussed in light of possible sites of entry for foliar applied nutrients.     

Anatomical and ultrastructural study of the pharyngeal bulb in Protodrilus (Polychaeta,Archiannelida) II. The stomodeal epithelium and its cuticle

The epidermal and stomodeal cuticles of Protodrilus are described then compared. The thin epidermal cuticle, the thickness of which is about the same over all the body, is characterized both by the absence of fibrils in its deepest part and by the extension of epidermal microvilli above the cuticle. The stomodeal cuticle, the thickness of which is as variable as that of the epithelium, presents two layers of fibrils comparable to the collagen fibrils described in the cuticle of other Annelida, as well as a relatively diversified supramicrovillous coating. The anterior cuticular thickening or grating plate, is characterized by the length of the epithelial microvilli, the thickness of the cuticular matrix and the superficial cuticular zone with supramicrovillous denticles supported by an axis of fibrous bundles. In the stomodeal cuticle, the fibrillar material seems to give to the cuticle a best resistance to deformation during the pharyngeal bulb contraction, while an especially elaborated supramicrovillous coating is found in regions most exposed to friction. These features contrast with the relative simplicity of the epidermal cuticle.     

Monitoring and visualising plant cuticles by confocal laser scanning microscopy

The present work shows the visualisation of phenolics and flavonoids of plant cuticles by confocal laser scanning microscopy (CLSM). Selected isolated fruit and leaf cuticles were monitored on the basis of autofluorescent phenolics and flavonoids which, in most cases, permitted us to obtain three-dimensional images of the cuticular membranes. The utility of this technique in investigations of cuticular translocation and diffusion of exogenous applied chemicals and cuticle degradation has also been explored.     

Transverse cuticular structures arise above actin filament bundles in the epidermis of Calpodes ethlius (Stöll) (Lepidoptera : Hesperiidae)

Rhodaminyl phalloin labelling of larval epidermal cells in Calpodes ethlius (Stöll) (Lepidoptera : Hesperiidae) shows dorsal areas with apical bundles of F-actin. The bundles are present only during the first 36 hr of the 5th stadium. Most cells have only one or 2, rarely 3, 4 or 5. The bundles extend into the overlying cuticle as the cores of large helical microvilli that continue on as transverse cuticle components, resembling very large helical pore canals. The transverse structures are like those seen in extensible insect cuticles that may allow cuticular stretching during larval growth. Neither the bundles nor the transverse structures are easily resolvable by conventional stains for LM or EM. The results suggest that transverse fibrillar structures may be a more common component of soft cuticles than has been generally realized.     

Cuticular Proteins in Insects and Crustaceans

Comparisons between crustacean and insect cuticles are hamperedby the paucity of cuticular protein sequences for the former.Sufficient complete sequences are available for insect cuticularproteins to allow recognition of conserved motifs and relationshipsamong proteins that reflect the type of cuticle from which theyhave been extracted. All five sequences from an arachnid andtwo of 14 from crustaceans have a motif found in the largestgroup of insect cuticular proteins. Numerous insights have beengained from studying insect cuticular proteins and their genes.These insights have been summarized in hopes of encouraginginterest in building on the foundations laid by Dorothy Skinnerwith the exoskeleton of Gecarcinus.     

Development of plant cuticles: fine structure and cutin composition of Clivia miniata Reg. leaves

The fine structure and monomeric composition of the ester-cutin fraction (susceptible to BF₃/CH₃OH transesterification) of the adaxial leaf cuticle of Clivia miniata Reg. were studied in relation to leaf and cuticle development. Clivia leaves grow at their base such that cuticle and tissues increase in age from the base to the tip. The zone of maximum growth (cell expansion) was located between 1 and 4 cm from the base. During cell expansion, the projected surface area of the upper epidermal cells increased by a factor of nine. In the growth region the cuticle consists mainly of a polylamellate cuticle proper of 100–250 nm thickness. After cell expansion has ceased both the outer epidermal wall and the cuticle increase in thickness. Thickening of the cuticle is accomplished by interposition of a cuticular layer between the cuticle proper and the cell wall. The cuticular layer exhibits a reticulate fine structure and contributes most of the total mass of the cuticle at positions above 6 cm from the leaf base. The composition of ester cutin changed with the age of cuticles. In depolymerisates from young cuticles, 26 different monomers could be detected whereas in older ones their number decreased to 13. At all developmental stages, 9,16-/10,16-dihydroxyhexadecanoic acid (positional isomers not separated), 18-hydroxy-9-octadecenoic acid, 9,10,18-trihydroxyoctadecanoic acid and 9,10-epoxy-18-hydroxyoctadecanoic acid were most frequent with the epoxy alkanoic acid clearly predominating (47% at 16 cm). The results are discussed as to (i) the age dependence of cutin composition, (ii) the relationship between fine structure and composition, (iii) the composition of the cuticle proper, the cuticular layer and the non-depolymerizable cutin fraction, and (iv) the polymeric structure of cutin.Abbreviations CL cuticular layer - CP cuticle proper - MX cutin polymer matrix     

Giant cuticular pores in Eidothea zoexylocarya (Proteaceae) leaves

Ubiquitous, large diameter pores have not previously been adequately demonstrated to occur in leaf cuticles. Here we show conclusively that such structures occur in Eidothea zoexylocarya, a rainforest tree species of Proteaceae restricted to the Australian Wet Tropics. The pores are abundant, large-diameter apertures (～1 μm), that extend perpendicularly most of the way through the cuticle from the inside. They occur on both sides of the leaf, but are absent from the cuticle associated with stomatal complexes on the abaxial side. No such pores were found in any other species, including the only other species of Eidothea, E. hardeniana from New South Wales, and other species that have previously been purported to possess cuticular pores. To determine whether these pores made the cuticles more leaky to water vapor, we measured astomatous cuticular conductances to water vapor for E. zoexylocarya and seven other Proteaceae species of the Wet Tropics. Cuticular conductance for E. zoexylocarya was relatively low, indicating that the prominent pores do not increase conductance. The function of the pores is currently obscure, but the presence of both pores and an adaxial hypodermis in E. zoexylocarya but not E. hardeniana suggests evolution in response to greater environmental stresses in the tropics.