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。     

植物角质层蜡质合成与调控的分子生物学研究进展

角质层覆盖于陆生植物的地上部分。沉积于其表面的外角质层蜡质组成了植物与外部环境之间的屏障。蜡质的合成是由大量酶类协同作用的结果,又是一个积极可调控的过程。综述了近年来角质层蜡质合成与调控的分子生物学研究进展,包括突变体筛选、基因克隆和鉴定,以及功能基因组学研究等三方面,并对植物蜡质代谢基因克隆鉴定中存在的问题进行了探讨。     

植物角质层基因研究进展

角质层是形成于陆生植物表皮细胞壁外表面的脂质保水层。角质层的基本功能是保水,同时也在响应逆境胁迫、自我清洁及器官发育等方面发挥作用。角质层通常由角质和蜡质组成。角质是角质层的主要结构成分,其主要组分是聚酯。蜡质成分主要为极长链饱和脂肪酸及其衍生物。这些组分在内质网上合成后被转运到细胞表面,进一步形成完整的角质层结构。近年来通过对角质层相关突变体及相应基因的研究,人们对角质层在合成、转运、形成及调控等各个阶段都有了较为深入的认识。蜡质和角质的合成途径已在角质层相关基因功能的解释下逐渐浮出水面。有关角质层前体转运方面的研究,主要的突破在于ABCG全转运蛋白的发现和功能解析。在角质层形成的机理方面,角质层基因中的酯酶和脂酶类基因的研究有助于进一步认识这个复杂的过程。在基因调控方面,新的转录因子基因和角质层与环境之间的相互关系研究,也为已知的调控网络增加了新内容。该文综述了目前关于角质层相关基因的最新研究进展。     

植物超长链脂肪酸及角质层蜡质生物合成相关酶基因研究现状

超长链脂肪酸(very long chain fatty acids, VLCFAs)在生物体中具有广泛的生理功能, 它们参与种子甘油酯、生物膜膜脂及鞘脂的合成, 并为角质层蜡质的生物合成提供前体物质。角质层是覆盖在植物地上部分最表层的保护层, 由角质和蜡质组成, 其中蜡质又分为角质层表皮蜡和内部蜡, 在植物生长发育、适应外界环境方面起重要作用。VLCFAs的合成由脂肪酰-CoA延长酶催化, 该酶是由b-酮脂酰-CoA合酶、b-酮脂酰-CoA还原酶、b-羟脂酰-CoA脱水酶和反式烯脂酰-CoA还原酶组成的多酶体系。合成后的VLCFAs通过脱羰基与酰基还原作用进入角质层蜡质合成途径, 形成各种蜡质组分。文章就VLCFAs及角质层蜡质合成代谢途径中相关酶基因研究进展方面做了综述, 并对植物蜡质基因研究中存在的问题提出一些看法。     

植物角质层蜡质基因的研究进展

角质层是覆盖在植物地上部分最表层的保护层,具有降低植物表面的水分散失、防止紫外线辐射伤害和抵抗病虫害侵入等环境胁迫等功能,在植物适应外界环境作用方面起重要作用.作对近年来角质层蜡质基因的研究进展进行综述,同时也对蜡质基因的研究前景提出一些看法.     

胡杨叶片表面角质提取方法的建立

以内蒙古额济纳地区的胡杨叶片为材料,建立了一种从角质层较厚的植物叶片中分离提取角质成分的方法。该方法能够最大限度除去胡杨叶片上的蜡质及其他脂类杂质成分干扰,结果准确,产率高,得到的角质可以直接用于气谱、质谱仪进行含量和成分的鉴定分析。     

植物表皮蜡质与抗旱及其分子生物学

植物表皮蜡质是覆盖在植物最外层的一类有机混合物总称,它是植物自我防护的最后一道屏障,在植物生长发育过程中起重要作用。文章就植物表皮蜡质的成分、转运、晶体的形成和发育、形态结构以及环境变化对表皮蜡质的影响,特别是在蜡质与角质蒸腾、叶片水分利用效率和产量的关系以及蜡质的生理作用和合成过程的分子生物学研究进展方面做了系统综述,并对植物蜡质研究中存在的问题做了探讨。     

花生种皮蜡质和角质层与黄曲霉侵染和产毒的关系

黄曲霉侵染花生的研究表明,种皮破损的黄曲霉毒素含量显著高于种皮完整的,种皮对黄曲霉侵染和产毒起着重要屏障作用。采用氯仿去除种皮蜡质,用KOH或角质酶去除种皮角质层后,种子黄曲霉感染率和黄曲霉毒素含量显著提高。种皮蜡质和角质层同时去除的与种皮破损的黄曲霉感染率和毒素含量差异不显著,表明种皮的抗性成份主要是蜡质和角质层。种皮蜡质含量测定和种皮表面扫描电镜观察表明,蜡质的含量和角质层的厚度与品种的抗性有关。抗性品种种皮蜡质含量显著高于感病品种。种皮蜡质提取物在体外抑菌效果不显著。说明蜡质的抗性作用主要是物理性阻止黄曲霉菌的穿透。     

植物角质层对非生物逆境胁迫响应研究进展

角质层,包括角质和蜡质,是主要由脂肪酸及其衍生物构成的覆盖在植物的外表面的高度疏水层,在植物生长发育过程中起到非常重要的保护屏障作用。除了在极端温度、干旱、高盐等多种非生物逆境胁迫下起到保护作用外,还能够保护植物内部组织免受细菌、真菌病原体的侵染。现就植物角质层的组成、合成途径以及与植物抗逆性,特别是与抗旱能力的关系方面的最新研究进展进行了综述。     

大豆Glyma03g24460基因功能预测及表达分析

大豆Glyma03g24460基因,与拟南芥ECERIFERUM1(CER1)基因具有高度的同源性,是一种植物角质层蜡质基因,参与植物角质层蜡质的合成。本研究对其进行基因功能预测和表达分析,利用Plant CARE分析启动子元件发现在基因启动子序列中含有黄酮合成、激素响应、生物和非生物胁迫相关的元件。氨基酸序列比对发现Glyma03g24460与其他物种的脂肪醛脱羧酶基因家族成员有很高的相似度。Glyma03g24460在大豆的q RT-PCR结果表明,它主要在植物的地上器官表达,并且可以受到ABA及干旱等非生物胁迫的诱导表达。     

Plant response to drought stress simulated by ABA application: Changes in chemical composition of cuticular waxes

Plant cuticles form the interface between epidermal plant cells and the atmosphere. The cuticle creates an effective barrier against water loss, bacterial and fungal infection and also protects plant tissue from UV radiation. It is composed of the cutin matrix and embedded soluble lipids also called waxes. Chemical composition of cuticular waxes and physiological properties of cuticles are affected by internal regulatory mechanisms and environmental conditions (e.g. drought, light, and humidity). Here, we tested the effect of drought stress simulation by the exogenous application of abscisic acid (ABA) on cuticular wax amount and composition. ABA-treated plants and control plants differed in total aboveground biomass, leaf area, stomatal density and aperture, and carbon isotope composition. They did not differ in total wax amount per area but there were peculiar differences in the abundance of particular components. ABA-treated plants contained significantly higher proportions of aliphatic components characterized by chain length larger than C₂₆, compared to control plants. This trend was consistent both between and within different functional groups of wax components. This can lead to a higher hydrophobicity of the cuticular transpiration barrier and thus decrease cuticular water loss in ABA-treated plants. At both ABA-treated and control plants alcohols with chain length C₂₄ and C₂₆ were predominant. Such a shift towards wax compounds having a higher average chain length under drought conditions can be interpreted as an adaptive response of plants towards drought stress.     

In situ analysis by microspectroscopy reveals triterpenoid compositional patterns within leaf cuticles of Prunus laurocerasus

The cuticular waxes on the leaves of Prunus laurocerasus are arranged in distinct layers differing in triterpenoid concentrations (Jetter et al., Plant Cell Environ 23:619–628, 2000). In addition to this transversal gradient, the lateral distribution of cuticular triterpenoids must be investigated to fully describe the spatial distribution of wax components on the leaf surfaces. In the present investigation, near infrared (NIR) Raman microspectroscopy, coherent anti-Stokes Raman scattering (CARS) microscopy, and third harmonic generation (THG) spectroscopy were employed to map the triterpenoid distribution in isolated cuticles from adaxial and abaxial sides of P. laurocerasus leaves. The relative concentrations of ursolic acid and oleanolic acid were calculated by treating the cuticle spectra as linear combinations of reference spectra from the major compounds found in the wax. Raman maps of the adaxial cuticle showed that the triterpenoids accumulate to relatively high concentrations over the periclinal regions of the pavement cells, while the very long chain aliphatic wax constituents are distributed fairly evenly across the entire adaxial cuticle. In the analysis of the abaxial cuticles, the triterpenoids were found to accumulate in greater amounts over the guard cells relative to the pavement cells. The very long chain aliphatic compounds accumulated in the cuticle above the anticlinal cell walls of the pavement cells, and were found at low concentrations above the periclinals and the guard cells.     

Leaf cuticular waxes are arranged in chemically and mechanically distinct layers: evidence from Prunus laurocerasus L.

The composition and spatial arrangement of cuticular waxes on the leaves of Prunus laurocerasus were investigated. In the wax mixture, the triterpenoids ursolic acid and oleanolic acid as well as alkanes, fatty acids, aldehydes, primary alcohols and alcohol acetates were identified. The surface extraction of upper and lower leaf surfaces yielded 280 mg m ^{? 2} and 830 mg m ^{? 2}, respectively. Protocols for the mechanical removal of waxes from the outermost layers of the cuticle were devised and evaluated. With the most selective of these methods, 130 mg m ^{? 2} of cuticular waxes could be removed from the adaxial surface before a sharp, physically resistant boundary was reached. Compounds thus obtained are interpreted as ‘epicuticular waxes’ with respect to their localization in a distinct layer on the surface of the cutin matrix. The epicuticular wax film can be transferred onto glass and visualized by scanning electron microscopy. Prunus laurocerasus epicuticular waxes consisted entirely of aliphatic compounds, whereas the remaining intracuticular waxes comprised 63% of triterpenoids. The ecological relevance of this layered structure for recognition by phytotrophic fungi and herbivorous insects that probe the surface composition for sign stimuli is discussed.     

Chemical composition of the Prunus laurocerasus leaf surface. Dynamic changes of the epicuticular wax film during leaf development

The seasonal development of adaxial Prunus laurocerasus leaf surfaces was studied using newly developed methods for the mechanical removal of epicuticular waxes. During epidermal cell expansion, more than 50 microg leaf(-1) of alkyl acetates accumulated within 10 d, forming an epicuticular wax film approximately 30 nm thick. Then, alcohols dominated for 18 d of leaf development, before alkanes accumulated in an epicuticular wax film with steadily increasing thickness (approximately 60 nm after 60 d), accompanied by small amounts of fatty acids, aldehydes, and alkyl esters. In contrast, the intracuticular waxes stayed fairly constant during development, being dominated by triterpenoids that could not be detected in the epicuticular waxes. The accumulation rates of all cuticular components are indicative for spontaneous segregation of intra- and epicuticular fractions during diffusional transport within the cuticle. This is the first report quantifying the loss of individual compound classes (acetates and alcohols) from the epicuticular wax mixture. Experiments with isolated epicuticular films showed that neither chemical conversion within the epicuticular film nor erosion/evaporation of wax constituents could account for this effect. Instead, transport of epicuticular compounds back into the tissue seems likely. Possible ecological and physiological functions of the coordinate changes in the composition of the plant surface layers are discussed.     

CUTICULAR LIPIDS OF TERRESTRIAL PLANTS AND ARTHROPODS: A COMPARISON OF THEIR STRUCTURE, COMPOSITION, AND WATERPROOFING FUNCTION

1. Lipids deposited on the surface or embedded within the cuticle of terrestrial plants and arthropods are primarily responsible for the observed low rates of water loss through the cuticle. 2. These lipids are a mixture of long-chain compounds which include hydrocarbons (saturated, unsaturated, branched), wax esters, free fatty acids, alcohols, ketones, aldehydes, and cyclic compounds. 3. The cuticle of both plants and arthropods is a continuous, non-cellular multilayered membrane which overlies the epidermal cells. 4. In arthropods, horizontal division of the cuticle into layers is clearly visible. In plants, the layers comprising the cuticle are not sharply demarcated. 5. The substance responsible for the structural integrity of the plant cuticle (cutin) is composed of cross-esterified fatty acids; structural integrity in arthropod cuticle is provided by a chitin-protein complex. 6. Cuticular lipids are probably formed near the surface in both plants and arthropods; however, specific sites of synthesis are known for only a few species and little is known about their transport from these sites to the surface. The elaborate pore canal and wax canal system of arthropod cuticle is absent from plants. 7. The physical structure and arrangement of the lipid deposits on the cuticular surface that are so important in controlling water movement depend on both quantity and chemical composition, and are, in turn, specific to each species in relation to its environment. 8. Different lipid components are not equally efficient in reducing transpiration. Maximum waterproofing effectiveness is provided by long-chain, saturated, non-polar molecules containing few methyl branches. 9. Plants and arthropods can, within genetic constraints, alter the composition of their cuticular waxes to improve impermeability when conditions require increased water conservation. 10. None of the models proposed to explain the change in arthropod cuticular permeability which occurs at a species-specific temperature (‘transition temperature’) in many species is supported by the experimental data now available.     

Localization of the Transpiration Barrier in the Epi- and Intracuticular Waxes of Eight Plant Species: Water Transport Resistances Are Associated with Fatty Acyl Rather Than Alicyclic Components

Plant cuticular waxes play a crucial role in limiting nonstomatal water loss. The goal of this study was to localize the transpiration barrier within the layered structure of cuticles of eight selected plant species and to put its physiological function into context with the chemical composition of the intracuticular and epicuticular wax layers. Four plant species (Tetrastigma voinierianum, Oreopanax guatemalensis, Monstera deliciosa, and Schefflera elegantissima) contained only very-long-chain fatty acid (VLCFA) derivatives such as alcohols, alkyl esters, aldehydes, and alkanes in their waxes. Even though the epicuticular and intracuticular waxes of these species had very similar compositions, only the intracuticular wax was important for the transpiration barrier. In contrast, four other species (Citrus aurantium, Euonymus japonica, Clusia flava, and Garcinia spicata) had waxes containing VLCFA derivatives, together with high percentages of alicyclic compounds (triterpenoids, steroids, or tocopherols) largely restricted to the intracuticular wax layer. In these species, both the epicuticular and intracuticular waxes contributed equally to the cuticular transpiration barrier. We conclude that the cuticular transpiration barrier is primarily formed by the intracuticular wax but that the epicuticular wax layer may also contribute to it, depending on species-specific cuticle composition. The barrier is associated mainly with VLCFA derivatives and less (if at all) with alicyclic wax constituents. The sealing properties of the epicuticular and intracuticular layers were not correlated with other characteristics, such as the absolute wax amounts and thicknesses of these layers.The plant cuticle is one of the major adaptations of vascular plants for life in the atmospheric environment. Accordingly, the primary function of cuticles is to limit nonstomatal water loss and, thus, to protect plants against drought stress (Burghardt and Riederer, 2006). However, plant cuticles also play roles in minimizing the adhesion of dust, pollen, and spores (Barthlott and Neinhuis, 1997), protecting tissues from UV radiation (Krauss et al., 1997; Solovchenko and Merzlyak, 2003), mediating biotic interactions with microbes (Carver and Gurr, 2006; Leveau, 2006; Hansjakob et al., 2010, 2011; Reisberg et al., 2012) as well as insects (Eigenbrode and Espelie, 1995; Müller and Riederer, 2005), and preventing deleterious fusions between different plant organs (Tanaka and Machida, 2013).Cuticles are composite (nonbilayer) membranes consisting of an insoluble polymer matrix and solvent-soluble waxes. The polymer matrix (MX) is mainly made of the hydroxy fatty acid polyester cutin (Nawrath, 2006) and also contains polysaccharides and proteins (Heredia, 2003). In contrast, cuticular waxes are complex mixtures of aliphatic compounds derived from very-long-chain fatty acids (VLCFAs) with hydrocarbon chains of C₂₀ and more (Jetter et al., 2007). Wax quantities and compositions vary greatly between plant species and, in many cases, even between organs and developmental stages. Diverse VLCFA derivatives can be present, including free fatty acids, aldehydes, ketones, primary and secondary alcohols, alkanes, and alkyl esters. Besides, the cuticular waxes of many plant species also contain cyclic compounds such as triterpenoids and aromatics.In order to characterize the physiological function of cuticular waxes, methods have been developed for the isolation of astomatous cuticles and the measurement of transpiration rates under exactly controlled conditions, so that well-defined physical transport parameters such as permeances and resistances can be determined and compared across species and organs (Schönherr and Lendzian, 1981; Kerstiens, 1996; Riederer and Schreiber, 2001; Lendzian, 2006). With these methods, it was demonstrated that the cuticular water permeance increases by up to 3 orders of magnitude upon wax removal, thus showing the central role of waxes as a transpiration barrier (Schönherr, 1976). Permeances for water determined so far with astomatous isolated leaf cuticular membranes (CMs) or in situ leaf cuticles range over 2.5 orders of magnitude, from 3.63 × 10⁻⁷ m s⁻¹ (Vanilla planifolia) to 7.7 × 10⁻⁵ m s⁻¹ (Maianthemum bifolium; Riederer and Schreiber, 2001).The species-dependent differences of both wax composition and permeance led to a search for correlations between cuticle structure and function. If such a structure-function relationship could be established, then it would become possible to select or alter wax composition in order to improve cuticle performance in crop species (Kosma and Jenks, 2007). However, all attempts to understand cuticle permeance based on cuticle composition have failed so far: correlations between wax amounts and permeances could not be established, contrary to the common assumption that thicker wax layers must provide better protection against desiccation (Schreiber and Riederer, 1996; Riederer and Schreiber, 2001). Similarly, a correlation between wax quality (i.e. the relative portions of its constituents) and permeance could also not be established to date (Burghardt and Riederer, 2006). It is not clear how certain wax components contribute to the vital barrier function of the cuticle.Previous attempts to establish wax structure-function relationships may have failed because only bulk wax properties were studied and important effects of substructures were averaged out. However, distinct compartments of wax exist within the cuticle, most prominently as a layer of intracuticular wax embedded within the MX and a layer of epicuticular wax deposited on the outer surface of the polymer (Jeffree, 2006). Over the last years, methods have been developed that allow the selective removal of epicuticular wax by adhesive surface stripping, followed by equally selective extraction of intracuticular wax (Jetter et al., 2000; Jetter and Schäffer, 2001). Chemical analyses showed that, for most plant species investigated to date, both wax layers have distinct compositions (Buschhaus and Jetter, 2011). The most pronounced differences between the layers were found for the triterpenoids, which were localized predominantly (or even exclusively) in the intracuticular wax. These findings raised the possibility that the chemically distinct wax layers might also have distinct functions, leading back to the long-standing question of whether the water barrier function is exerted by the intracuticular and/or the epicuticular wax. There are only scant data to answer this question so far, mainly because methods allowing a distinction between epicuticular and intracuticular waxes were established only recently. Using these sampling techniques, it was recently found that, for leaves of Prunus laurocerasus, the epicuticular wax layer does not contribute to the transpiration barrier (Zeisler and Schreiber, 2016). In contrast, it had been reported that removal of the epicuticular wax layer from tomato (Solanum lycopersicum) fruit caused an approximately 2-fold increase in transpiration, suggesting that, in this species, the epicuticular layer constitutes an important part of the barrier (Vogg et al., 2004). Based on these conflicting reports, it is not clear to what extent the intracuticular or the epicuticular waxes contribute to the sealing function of the plant skin.The goal of this study was to localize the transpiration barrier within the cuticular membrane of selected plant species and to put the physiological function into context with the chemical composition of both the epicuticular and intracuticular wax layers. To this end, we selected eight species from which leaf cuticles could be isolated and methods for step-wise wax removal could be applied without damaging the cuticle. Preliminary studies had shown that the adaxial cuticles on leaves of Citrus aurantium (Rutaceae), Euonymus japonica (Celastraceae), Clusia flava (Clusiaceae), Garcinia spicata (Clusiaceae), Tetrastigma voinierianum (Vitaceae), Oreopanax guatemalensis (Araliaceae), Monstera deliciosa (Araceae), and Schefflera elegantissima (Araliaceae) were astomateous and showed wide chemical diversity. Therefore, these eight species were selected to address the following questions: (1) What are the amounts of epicuticular and intracuticular waxes? (2) Do compositional differences exist between the layers? (3) Where are the cuticular triterpenoids located? (4) How much do the epicuticular and intracuticular waxes contribute to the transpiration barrier? (5) Is the barrier associated with certain components of the intracuticular or epicuticular waxes?     

The fruit cuticles of wild tomato species exhibit architectural and chemical diversity, providing a new model for studying the evolution of cuticle function

The cuticle covers the aerial epidermis of land plants and plays a primary role in water regulation and protection from external stresses. Remarkable species diversity in the structure and composition of its components, cutin and wax, have been catalogued, but few functional or genetic correlations have emerged. Tomato (Solanum lycopersicum) is part of a complex of closely related wild species endemic to the northern Andes and the Galapagos Islands (Solanum Sect. Lycopersicon). Although sharing an ancestor <7 million years ago, these species are found in diverse environments and are subject to unique selective pressures. Furthermore, they are genetically tractable, since they can be crossed with S. lycopersicum, which has a sequenced genome. With the aim of evaluating the relationships between evolution, structure and function of the cuticle, we characterized the morphological and chemical diversity of fruit cuticles of seven species from Solanum Sect. Lycopersicon. Striking differences in cuticular architecture and quantities of cutin and waxes were observed, with the wax coverage of wild species exceeding that of S. lycopersicum by up to seven fold. Wax composition varied in the occurrence of wax esters and triterpenoid isomers. Using a Solanum habrochaites introgression line population, we mapped triterpenoid differences to a genomic region that includes two S. lycopersicum triterpene synthases. Based on known metabolic pathways for acyl wax compounds, hypotheses are discussed to explain the appearance of wax esters with atypical chain lengths. These results establish a model system for understanding the ecological and evolutionary functional genomics of plant cuticles.     

Arabidopsis LTPG Is a Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface

Plant epidermal cells dedicate more than half of their lipid metabolism to the synthesis of cuticular lipids, which seal and protect the plant shoot. The cuticle is made up of a cutin polymer and waxes, diverse hydrophobic compounds including very-long-chain fatty acids and their derivatives. How such hydrophobic compounds are exported to the cuticle, especially through the hydrophilic plant cell wall, is not known. By performing a reverse genetic screen, we have identified LTPG, a glycosylphosphatidylinositol-anchored lipid transfer protein that is highly expressed in the epidermis during cuticle biosynthesis in Arabidopsis thaliana inflorescence stems. Mutant plant lines with decreased LTPG expression had reduced wax load on the stem surface, showing that LTPG is involved either directly or indirectly in cuticular lipid deposition. In vitro 2-p-toluidinonaphthalene-6-sulfonate assays showed that recombinant LTPG has the capacity to bind to this lipid probe. LTPG was primarily localized to the plasma membrane on all faces of stem epidermal cells in the growing regions of inflorescence stems where wax is actively secreted. These data suggest that LTPG may function as a component of the cuticular lipid export machinery.     

Ontogenetic variation in chemical and physical characteristics of adaxial apple leaf surfaces

The reaction of plants to environmental factors often varies with developmental stage. It was hypothesized, that also the cuticle, the outer surface layer of plants is modified during ontogenesis. Apple plantlets, cv. Golden Delicious, were grown under controlled conditions avoiding biotic and abiotic stress factors. The cuticular wax surface of adaxial apple leaves was analyzed for its chemical composition as well as for its micromorphology and hydrophobicity just after unfolding of leaves ending in the seventh leaf insertion. The outer surface of apple leaves was formed by a thin amorphous layer of epicuticular waxes. Epidermal cells of young leaves exhibited a distinctive curvature of the periclinal cell walls resulting in an undulated surface of the cuticle including pronounced lamellae, with the highest density at the centre of cells. As epidermal cells expanded during ontogenesis, the upper surface showed only minor surface sculpturing and a decrease in lamellae. With increasing leaf age the hydrophobicity of adaxial leaf side decreased significantly indicated by a decrease in contact angle. Extracted from plants, the amount of apolar cuticular wax per area unit ranged from only 0.9 microgcm(-2) for the oldest studied leaf to 1.5 microgcm(-2) for the youngest studied leaf. Differences in the total amount of cuticular waxes per leaf were not significant for older leaves. For young leaves, triterpenes (ursolic acid and oleanolic acid), esters and alcohols were the main wax components. During ontogenesis, the proportion of triterpenes in total mass of apolar waxes decreased from 32% (leaf 1) to 13% (leaf 7); absolute amounts decreased by more than 50%. The proportion of wax alcohols and esters, and alkanes to a lesser degree, increased with leaf age, whereas the proportion of acids decreased. The epicuticular wax layer also contained alpha-tocopherol described for the first time to be present also in the epicuticular wax. The modifications in the chemical composition of cuticular waxes are discussed in relation to the varying physical characteristics of the cuticle during ontogenesis of apple leaves.