Resistance in sorghum to the shoot fly, Atherigona soccata: epicuticular wax and wetness of the central whorl leaf of young seedlings

Sorghum genotypes known to be resistant or susceptible to shoot fly, Atherigona soccata Rondani were examined by scanning electron microscopy for differences in epicuticular wax structure and wetness of the central leaf whorl. Two major types of wax structures were observed: shoot fly resistant and moderately resistant genotypes were characterised by a smooth amorphous wax layer and sparse wax crystals while susceptible genotypes possessed a dense meshwork of crystalline epicuticular wax. The density of wax crystals decreased from the third leaf to the seventh leaf stage and was related to both seedling age and leaf position. Water droplets on susceptible genotypes with dense wax crystals showed spreading at the edges indicating a tendency to wet easily. In resistant genotypes with less dense wax crystals the droplets remained intact and did not spread.     

Epicuticular wax crystals of Wollemia nobilis: morphology and chemical composition

BACKGROUND AND AIMS: The morphology of the epicuticular leaf waxes of Wollemia nobilis (Araucariaceae) was studied with special emphasis on the relationship between the microstructure of epicuticular wax crystals and their chemical composition. Wollemia nobilis is a unique coniferous tree of the family Araucariaceae and is of very high scientific value as it is the sole living representative of an ancient genus, which until 1994 was known only from fossils. METHODS: Scanning electron microscopy (SEM), gas chromatography (GC) combined with mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR) were used for characterizing the morphology and the chemical structure of the epicuticular wax layer of W. nobilis needles. KEY RESULTS: The main component of the leaf epicuticular wax of W. nobilis is nonacosan-10-ol. This secondary alcohol together with nonacosane diols is responsible for the tubular habit of the epicuticular wax crystals. Scanning electron micrographs revealed differences in the fine structure of adaxial and abaxial leaf surfaces that could be explained by gas chromatographic studies after selective mechanical removal of the waxes. CONCLUSIONS: SEM investigations established the tubular crystalline microstructure of the epicuticular wax of W. nobilis leaves. GC-MS and NMR experiments showed that nonacosan-10-ol is the major constituent of the epicuticular wax of W. nobilis leaves.     

Epicuticular wax structures on stems and comparison between stems and leaves – A survey

The cuticle, forming the outermost layer of plant tissues and being in direct contact with the environment, consists of waxes and cutin. Waxes are hydrophobic substances that are divided in two groups: intra- and epicuticular, depending on their localisation. Epicuticular waxes appear as smooth coverings, however, many plants also produce superimposed wax structures of a crystalline nature. While studies of waxes have almost exclusively focused on leaves, here a survey of epicuticular wax structures on stems is presented. The stem surface of 343 higher plant taxa, representing 80 families, was examined using scanning electron microscopy. The adaxial and abaxial surfaces of leaves of 319 taxa were also examined to determine the relationship between wax structures on stems and leaves. Wax structures are classified, described and discussed. The results of the study indicate that stems exhibit the same main wax crystal types that have been described for leaves. Seventy percent of the examined taxa produced wax crystals on their stems. In ∼24% of the taxa, wax crystals were absent on leaves and found only on stems. In plant taxa that produce wax crystals, 40% exhibit the same type on either side of their leaves and on their stem. However, a much stronger morphological similarity exists between crystal shapes present on the adaxial and abaxial surfaces of leaves than between those present on the stem and those on leaves. In general, these observations suggest that stems are quite different than leaves in terms of their epicuticular wax structures.     

Structural analysis of wheat wax (Triticum aestivum, c.v. ‘Naturastar’ L.): from the molecular level to three dimensional crystals

In order to elucidate the self assembly process of plant epicuticular waxes, and the molecular arrangement within the crystals, re-crystallisation of wax platelets was studied on biological and non-biological surfaces. Wax platelets were extracted from the leaf blades of wheat (Triticum aestivum L., c.v. ‘Naturastar’, Poaceae). Waxes were analysed by gas chromatography (GC) and mass spectrometry (MS). Octacosan-1-ol was found to be the most abundant chemical component of the wax mixture (66 m%) and also the determining compound for the shape of the wax platelets. The electron diffraction pattern showed that both the wax mixture and pure octacosan-1-ol are crystalline. The re-crystallisation of the natural wax mixture and the pure octacosan-1-ol were studied by scanning tunnelling microscopy (STM), atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Crystallisation of wheat waxes and pure octacosano-1-ol on the non polar highly ordered pyrolytic graphite (HOPG) led to the formation of platelet structures similar to those found on the plant surface. In contrast, irregular wax morphologies and flat lying plates were formed on glass, silicon, salt crystals (NaCl) and mica surfaces. Movement of wheat wax through isolated Convallaria majalis cuticles led to typical wax platelets of wheat, arranged in the complex patterns typical for C. majalis. STM of pure octacosan-1-ol monolayers on HOPG showed that the arrangement of the molecules strictly followed the hexagonal structure of the substrate crystal. Re-crystallisation of wheat waxes on non-polar crystalline HOPG substrate showed that technical surfaces could be used to generate microstructured, biomimetic surfaces. AFM and SEM studies proved that a template effect of the substrate determined the orientation of the re-grown crystals. These effects of the structure and polarity of the substrate on the morphology of the epicuticular waxes are relevant for understanding interactions between biological as well as technical surfaces and waxes.     

Movement and regeneration of epicuticular waxes through plant cuticles

Regeneration of plant epicuticular waxes was studied in 24 plant species by high-resolution scanning electron microscopy. According to their regeneration behaviour, four groups could be distinguished: (i) regeneration occurs at all stages of development; (ii) regeneration occurs only during leaf expansion; (iii) regeneration occurs only in fully developed leaves; (iv) plants were not able to regenerate wax. Wax was removed from the leaves with water-based glue and a liquid polymer, i.e. water-based polyurethane dispersion. In young leaves these coverings could not be removed without damaging the leaves. After a few days, waxes appeared on the surface of these polymer films, which still adhered to the leaves. It is concluded that waxes move through the cuticle in a process similar to steam distillation. This hypothesis could be further substantiated in refined in vitro experiments. Wax isolated from Eucalyptus globulus was applied to a filter paper, subsequently covered with a liquid polymer and fixed onto a diffusion chamber filled with water. The diffusion chamber was put into a desiccator. After 8-10 days at room temperature, crystals similar in dimensions and shape to in situ crystals appeared on the surface of the polyurethane film. This indicates that waxes in molecular dimensions move together with the water vapor that permeates through the polymer membrane. Based on these results, we propose a new and simple hypothesis for the mechanism of wax movement: the molecules that finally form the epicuticular wax crystals are moved in the cuticular water current.     

Chemical composition of the epicuticular and intracuticular wax layers on the adaxial side of Ligustrum vulgare leaves

Previous research has shown that cuticular triterpenoids are exclusively found in the intracuticular wax layer of Prunus laurocerasus. To investigate whether this partitioning was species-specific, the intra- and epicuticular waxes were identified and quantified for the glossy leaves of Ligustrum vulgare, an unrelated shrub with similar wax morphology. Epicuticular wax was mechanically stripped from the adaxial leaf surface using the adhesive gum arabic. Subsequently, the organic solvent chloroform was used to extract the intracuticular wax from within the cutin matrix. The isolated waxes were quantified using gas chromatography with flame ionization detection and identified by mass spectrometry. The results were visually confirmed by scanning electron microscopy. The outer wax layer consisted entirely of homologous series of very-long-chain aliphatic compound classes. By contrast, the inner wax layer was dominated (80%) by two cyclic triterpenoids, ursolic and oleanolic acid. The accumulation of triterpenoids in the intracuticular leaf wax of a second, unrelated species suggests that this localization may be a more general phenomenon in smooth cuticles lacking epicuticular wax crystals. The mechanism and possible ecological or physiological reasons for this separation are currently being investigated.     

Effect of epicuticular wax crystals on the localization of artificially deposited sub-micron carbon-based aerosols on needles of <Emphasis Type="Italic">Cryptomeria japonica</Emphasis>

Elucidation of the mechanism of adsorption of particles suspended in the gas-phase (aerosol) to the outer surfaces of leaves provides useful information for understanding the mechanisms of the effect of aerosol particles on the growth and physiological functions of trees. In the present study, we examined the localization of artificially deposited sub-micron-sized carbon-based particles on the surfaces of needles of Cryptomeria japonica, a typical Japanese coniferous tree species, by field-emission scanning electron microscopy. The clusters (aggregates) of carbon-based particles were deposited on the needle surface regions where epicuticular wax crystals were sparsely distributed. By contrast, no clusters of the particles were found on the needle surface regions with dense distribution of epicuticular wax crystals. Number of clusters of carbon-based particles per unit area showed statistically significant differences between regions with sparse epicuticular wax crystals and those with dense epicuticular wax crystals. These results suggest that epicuticular wax crystals affect distribution of carbon-based particles on needles. Therefore, densely distributed epicuticular wax crystals might prevent the deposition of sub-micron-sized carbon-based particles on the surfaces of needles of Cryptomeria japonica to retain the function of stomata.     

Composition differences between epicuticular and intracuticular wax substructures: how do plants seal their epidermal surfaces?

The protective wax coating on plant surfaces has long been considered to be non-uniform in composition at a subcellular scale. In recent years, direct evidence has started to accumulate showing quantitative compositional differences between the epicuticular wax (i.e. wax exterior to cutin that can be mechanically peeled off) and intracuticular wax (i.e. wax residing within the mechanically resistant layer of cutin) layers in particular. This review provides a first synthesis of the results acquired for all the species investigated to date in order to assign chemical information directly to cuticle substructures, together with an overview of the methods used and a discussion of possible mechanisms and biological functions. The development of methods to probe the wax for z-direction heterogeneity began with differential solvent extractions. Further research employing mechanical wax removal by adhesives permitted the separation and analysis of the epicuticular and intracuticular wax. In wild-type plants, the intracuticular (1-30 μg cm(-2)) plus the epicuticular wax (5-30 μg cm(-2)) combined to a total of 8-40 μg cm(-2). Cyclic wax constituents, such as triterpenoids and alkylresorcinols, preferentially or entirely accumulate within the intracuticular layer. Within the very-long-chain aliphatic wax components, primary alcohols tend to accumulate to higher percentages in the intracuticular wax layer, while free fatty acids and alkanes in many cases accumulate in the epicuticular layer. Compounds with different chain lengths are typically distributed evenly between the layers. The mechanism causing the fractionation remains to be elucidated but it seems plausible that it involves, at least in part, spontaneous partitioning due to the physico-chemical properties of the wax compounds and interactions with the intracuticular polymers. The arrangement of compounds probably directly influences cuticular functions.     

Chemical composition of epicuticular wax crystals on the slippery zone in pitchers of five <Emphasis Type="Italic">Nepenthes</Emphasis> species and hybrids

Plants of the carnivorous genus Nepenthes efficiently trap insects in leaf pitchers, mostly employing epicuticular wax crystals on the pitcher walls to make them slippery for the prey. In the present study, the compositions and micromorphologies of the wax crystals of five Nepenthes species and hybrids were analysed in order to test whether the chemical principles underlying this ecological function are widespread within the genus. Three wax layers could be distinguished within the Nepenthes pitcher cuticles: (1) the outermost part of the crystals forming the platelets visible in standard scanning electron microscopy, (2) the bottom portion of the epicuticular wax crystals, and (3) an intracuticular wax layer. The composition of the intracuticular wax differed significantly from that of the neighbouring epicuticular layer. The compositions of corresponding wax mixtures from all five Nepenthes species and hybrids were very similar, with almost equal amounts of very long chain aldehydes and primary alcohols. While triacontanal (C₃₀ aldehyde) was prevailing in the epicuticular crystals of Nepenthes albomarginata and Nepenthes x intermedia, Nepenthes x superba and Nepenthes x henriana were found to have especially high percentages of dotriacontanal (C₃₂ aldehyde). Nepenthes “khasiana” had an intermediate aldehyde composition with almost equal amounts of both chain lengths.     

Chemical composition of the epicuticular and intracuticular wax layers on adaxial sides of Rosa canina leaves

BACKGROUND AND AIMS: The waxy cuticle is the first point of contact for many herbivorous and pathogenic organisms on rose plants. Previous studies have reported the average composition of the combined wax extract from both sides of rose leaves. Recently, the compositions of the waxes on the adaxial and abaxial surfaces of Rosa canina leaves were determined separately. In this paper, a first report is made on the compositions of the epicuticular and intracuticular wax layers of Rosa canina leaves. The methods described enable the determination of which compounds are truly available at the surface for plant-organism interactions. METHODS: An adhesive was used to mechanically strip the epicuticular wax from the adaxial leaf surface and the removal was visually confirmed using scanning electron microscopy. After the epicuticular wax had been removed, the intracuticular wax was then isolated using standard chemical extraction. Gas chromatography, flame ionization detection and mass spectrometry were used to identify and quantify compounds in the separated wax mixtures. KEY RESULTS: The epicuticular wax contained higher concentrations of alkanes and alkyl esters but lower concentrations of primary alcohols and alkenols when compared to the intracuticular wax. In addition, the average chain lengths of these compound classes were higher in the epicuticular wax. Secondary alcohols were found only in the epicuticular layer while triterpenoids were restricted mainly to the intracuticular wax. CONCLUSIONS: A gradient exists between the composition of the epi- and intracuticular wax layers of Rosa canina leaves. This gradient may result from polarity differences, in part caused by differences in chain lengths. The outer wax layer accessible to the phyllosphere showed a unique composition of wax compounds. The ecological consequences from such a gradient may now be probed.     

Physical, chemical and spectral properties of leaf surfaces of Berberis aquifolium (Pursh.)

Abstract The leaves of Berberis aquifolium (Pursh.) exhibit either diffuse or specular (shiny) reflection, depending on the variety, but in no case are the leaves obviously glaucous. The dull-surfaced leaves were less wettable than the glossy ones. Using scanning electron microscopy it was determined that the diffuse reflection was due to tubular crystals of wax 250 nm in diameter. The crystals were primarily composed of 19-nonacosanol, a 29-carbon secondary alcohol, as determined by gas chromatography-mass spectrometry. The chemical constituents of the wax underlying the tubes appeared to be the same as those of the wax from glossy leaves, with 29-carbon and 31-carbon n-alkanes and n-heptacosanol as major constituents. The reflection spectra of dull-surfaced (diffuse reflection) or glossy (specular reflection) leaves were the same, as were those of leaves with different amounts of epicuticular wax. Removing the epicuticular wax with chloroform did not change the spectrum.     

Epicuticular Wax and Cuticular Resistance in Rice

High leaf cuticular resistance has been reported as a component adaptation of plants to drought prone regions, Experiments were conducted to evaluate and characterize the role of epicuticular wax as a component of cuticular resistance to water vapor loss from rice (Oryza sativa L.) leaves. This information is necessary to determine the applicability of including higher cuticular resistance in an upland rice breeding program and to evaluate potential selection methods. Diffusion porometry, electron microscopy, and gas liquid chromatography were employed. Measurement of cuticular resistance by leaf diffusive resistance porometry after stomatal closure by exposure of rice leaves to pure CO₂ for 15 min was found sufficient to induce complete stomatal closure regardless of light level, and was superior to dark acclimation for this purpose. Removal of epicuticular wax from rice leaves by chloroform dip significantly reduced the cuticular resistance. Stressed plants were observed to increase cuticular resistance, illustrating the responsive nature of this characteristic. Gas liquid chromatography (GLC) of the chloroform leaf dip proved to be an expedient method of characterizing both quantitative and qualitative differences in the epicuticular wax of rice cullivars. The porometry and GLC techniques may be useful in selecting parents, spot checking in a pedigree program, or checking lines reaching the yield testing stage, but ate not well suited lor mass screening early generation progeny. Significant differences were found in the two rice cultivurs, ‘63–83′, an upland adapted rice from West Africa, and “IR20′, bred and selected in submerged paddy culture in ihc Philippines, by tlie above methods of characterizing cuticular resistance and epicuticular wax. These results are of ecological significance to plant breeders.     

What do microbes encounter at the plant surface? Chemical composition of pea leaf cuticular waxes

In the cuticular wax mixtures from leaves of pea (Pisum sativum) cv Avanta, cv Lincoln, and cv Maiperle, more than 70 individual compounds were identified. The adaxial wax was characterized by very high amounts of primary alcohols (71%), while the abaxial wax consisted mainly of alkanes (73%). An aqueous adhesive of gum arabic was employed to selectively sample the epicuticular wax layer on pea leaves and hence to analyze the composition of epicuticular crystals exposed at the outermost surface of leaves. The epicuticular layer was found to contain 74% and 83% of the total wax on adaxial and abaxial surfaces, respectively. The platelet-shaped crystals on the adaxial leaf surface consisted of a mixture dominated by hexacosanol, accompanied by substantial amounts of octacosanol and hentriacontane. In contrast, the ribbon-shaped wax crystals on the abaxial surface consisted mainly of hentriacontane (63%), with approximately 5% each of hexacosanol and octacosanol being present. Based on this detailed chemical analysis of the wax exposed at the leaf surface, their importance for early events in the interaction with host-specific pathogenic fungi can now be evaluated. On adaxial surfaces, approximately 80% of Erysiphe pisi spores germinated and 70% differentiated appressoria. In contrast, significantly lower germination efficiencies (57%) and appressoria formation rates (49%) were found for abaxial surfaces. In conclusion, the influence of the physical structure and the chemical composition of the host surface, and especially of epicuticular leaf waxes, on the prepenetration processes of biotrophic fungi is discussed.     

Wax-deficient anther1 is involved in cuticle and wax production in rice anther walls and is required for pollen development

In vegetative leaf tissues, cuticles including cuticular waxes are important for protection against nonstomatal water loss and pathogen infection as well as for adaptations to environmental stress. However, their roles in the anther wall are rarely studied. The innermost layer of the anther wall (the tapetum) is essential for generating male gametes. Here, we report the characterization of a T-DNA insertional mutant in the Wax-deficient anther1 (Wda1) gene of rice (Oryza sativa), which shows significant defects in the biosynthesis of very-long-chain fatty acids in both layers. This gene is strongly expressed in the epidermal cells of anthers. Scanning electron microscopy analyses showed that epicuticular wax crystals were absent in the outer layer of the anther and that microspore development was severely retarded and finally disrupted as a result of defective pollen exine formation in the mutant anthers. These biochemical and developmental defects in tapetum found in wda1 mutants are earlier events than those in other male-sterile mutants, which showed defects of lipidic molecules in exine. Our findings provide new insights into the biochemical and developmental aspects of the role of waxes in microspore exine development in the tapetum as well as the role of epicuticular waxes in anther expansion.     

Plant epicuticular lipids: alteration by herbicidal carbamates

The effect of several carbamates and trichloroacetic acid on the biosynthesis of epicuticular lipids from leaves of pea (Pisum sativum) was tested by chemical and visual methods. The carbamates tested included S-(2,3-dichloroallyl) diisopropylthiocarbamate (diallate), N-(3-chlorophenyl) isopropylcarbamate (chloropropham), S-ethyl dipropylthiocarbamate, and 2-chloroallyl diethyldithiocarbamate. Diallate reduced epicuticular lipids by 50% when the plants were root-treated and by 80% when vapor-treated. These results were supported by scanning electron microscopy and carbon replica techniques with transmission electron microscopy. The ratio of wax lipid components in the diallate-treated plants remained unchanged, with the exception of the primary alcohols, which were reduced. Diallate appears to interfere with the biosynthesis of a precursor to the elongation-decarboxylation pathway of lipid synthesis. N-(3-Chlorophenyl)isopropylcarbamate had no significant effect on total amounts of extractable epicuticular lipids, nor did it alter the structure of the wax formation on the leaves. The scanning electron microscopy micrographs indicated that S-ethyl dipropylthiocarbamate significantly reduced wax formation on pea leaves. 2-Chloroallyl diethyldithiocarbamate altered the structure of the wax formations, but not the total amount of wax (scanning electron microscopy). Trichloroacetic acid had little effect on wax deposition compared to diallate or S-ethyl dipropylthiocarbamate (scanning electron microscopy). The implication of the effect of the carbamates on epicuticular lipids and penetration of subsequent topically applied chemicals is discussed.     

Slippery surfaces of carnivorous plants: composition of epicuticular wax crystals in<Emphasis Type="Italic"> Nepenthes alata</Emphasis> Blanco pitchers

Plants in the genus Nepenthes obtain a substantial nutrient supply by trapping insects in highly modified leaves. A broad zone of the inner surface of these pitchers is densely covered with wax crystals on which most insects lose their footing. This slippery wax surface, capturing prey and preventing its escape from the trap, plays a pivotal role in the carnivorous syndrome. To understand the mechanism of slipperiness, the present investigation aimed at an ultrastructural and physico-chemical characterization of the wax crystals in pitchers of N. alata Blanco. Scanning electron microscopy revealed that entire platelets protruded perpendicularly from the surface. Methods were developed that allowed the mechanical removal of wax crystals from the pitcher surface. It could be shown that the sampling was selective for the epicuticular wax, relevant for plant–insect interactions. The crystals consisted of a mixture of aliphatic compounds dominated by very-long-chain aldehydes. Triacontanal, at 43% the most abundant constituent, was largely responsible for crystal formation. Solubility data indicate that the Nepenthes crystals contained polymeric forms of this aldehyde. The resulting mechanical properties of the polymer crystals and the mechanism of slipperiness are discussed.Abbreviation SEM scanning electron microscopy     

植物角质层内外蜡质的差异及其与抗逆性的关系

植物角质层是覆盖在植物地上部分的叶、花和非木质茎等器官表面的保护层,包括角质和蜡质。其中蜡质根据分布位置不同又分为表皮蜡质和内部蜡质。大量研究表明,表皮蜡质含量和结构在植物生长发育和抗逆性申发挥着重要作用。近年来有研究发现构成蜡质的成分在内外蜡质层中的分布存在差异,角质层蜡质成分影响植物抗逆性。本文针对角质层结构和内外蜡质差异性以及角质层结构和组成与植物抗逆性之间的关系进行了综述。     

The cuticle of the Buxbaumia viridis sporophyte

The sporophytes of Buxbaumia viridis are covered with a cuticle-like skin, which is peeling off when the capsules are ripe. Scanning electron microscopy (SEM) analysis showed that the supposed peeling of the cuticle of ripe capsules is not restricted to the cuticle, but to the complete epidermis. Additionally the SEM investigations showed that the capsule epidermis is covered by epicuticular wax crystals in different morphologies. Wax crystal morphologies indicate the presence of massive wax layers with small embedded and superimposed platelets and granules on top. For bryophytes such wax crystals were so far reported only from the Polytrichales, but they are common wax types of various groups of several phanerogams. Based on these micro-morphological markers the phylogenetic relationship between the cuticles of moss sporophytes and tracheophytes is discussed here.     

Relationship between selected morphological, anatomical and cytological characteristics of leaves and the level of tolerance to herbicides in strawberry cultivars

Foliar application of a mixture of herbicides containing phenmedipham, desmedipham and ethofumesate to the plants of nine strawberry cultivars revealed that there were differences in the level of plant tolerance to the applied chemicals. Light, polarized light and scanning electron microscopy were used to explain differences in tolerance to herbicides. The surface of strawberry leaves and cells was examined for stomata, hairs, trichomes, surface structures, cucticle, vacuole and oxalate crystals. The thicker the cuticle on the adaxial leaf surface, the thicker the layer of epicuticular waxes, greater number of large vacuoles and greater number of calcium oxalate crystals in epidermis cells were characteristic for cultivars with very good tolerance to herbicides. The cracking of epicuticular waxes layer was typical to cultivars with respectively low tolerance to herbicides.     

Structural Response of Soybean Leaf to Elevated CO２ Concentration

The effects of CO2 concentration on the morphological and anatomical characters of soybean (Glycine max) leaf were investigated by means of light microscopy and SEM. It was noticed that exomorphology did not show dramatic change, while stomatal density decreased with increasing CO2 concentration. Under SEM, no epicuticular wax was observed on both abaxial and adaxial sides of the control group as well as on adaxial side of the treatment group. However, leaf surface of abaxial side was noticed to be densely covered with microasterisk epicuticular wax when they were exposed to CO2-enriched environment. The epicuticular wax deposition was present in equal abundance on both stomatal and nonstomatal areas. Furthermore, leaf thickness increased significantly due largely to the origin of an extra layer of palisade in the treatment group. The results confirmed that CO2 enrichment might enhance cell division and induce greater quantity of epicuticular wax.