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.     

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.     

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.     

Two sides of a leaf blade: <Emphasis Type="Italic">Blumeria graminis</Emphasis> needs chemical cues in cuticular waxes of <Emphasis Type="Italic">Lolium perenne</Emphasis> for germination and differentiation

Plant surface characteristics were repeatedly shown to play a pivotal role in plant–pathogen interactions. The abaxial leaf surface of perennial ryegrass (Lolium perenne) is extremely glossy and wettable compared to the glaucous and more hydrophobic adaxial surface. Earlier investigations have demonstrated that the abaxial leaf surface was rarely infected by powdery mildew (Blumeria graminis), even when the adaxial surface was densely colonized. This led to the assumption that components of the abaxial epicuticular leaf wax might contribute to the observed impairment of growth and development of B. graminis conidia on abaxial surfaces of L. perenne. To re-assess this hypothesis, we analyzed abundance and chemical composition of L. perenne ab- and adaxial epicuticular wax fractions. While the adaxial epicuticular waxes were dominated by primary alcohols and esters, the abaxial fraction was mainly composed of n-alkanes and aldehydes. However, the major germination and differentiation inducing compound, the C₂₆-aldehyde n-hexacosanal, was not present in the abaxial epicuticular waxes. Spiking of isolated abaxial epicuticular Lolium waxes with synthetically produced n-hexacosanal allowed reconstituting germination and differentiation rates of B. graminis in an in vitro germination assay using wax-coated glass slides. Hence, the absence of the C₂₆-aldehyde from the abaxial surface in combination with a distinctly reduced surface hydrophobicity appears to be primarily responsible for the failure of normal germling development of B. graminis on the abaxial leaf surfaces of L. perenne.     

Cuticle micromorphology of Saxegothaea (Podocarpaceae)

The cuticle micromorphology of the leaves of the monospecific genus Saxegothaea (Podocarpaceae) was studied by scanning electron microscopy. The external and internal features of the adaxial and abaxial surfaces were characterized. The leaves are hypostomatic. The external adaxial cuticle is rugose with irregular ridges and shallow trench‐like structures that do not correspond to any feature of the inner cuticle surface. The external abaxial cuticle has densely crowded stomata arranged in two bands. The stomata are sunken with pronounced, interrupted Florin rings. Stomatal plugs were not observed. Internally, the adaxial epidermal cells are usually rectangular to square; the abaxial epidermal cells are mainly restricted to the midrib and margins and narrowly rectangular; any among the stomata are irregularly shaped. The stomata are nearly all in direct contact. They show unusual features, including an extra pair of cuticular flanges between the guard cell flanges and those of the lateral subsidiary cells, and ‘bridges’ of lateral subsidiary cell tissue extending polewards above the polar extensions to unite with those at their tips. Neither of these features has been reported previously in Podocarpaceae. The results are discussed in the light of recent phylogenetic studies. It is concluded that, despite its unique cuticular features, Saxegothaea should continue to be regarded as a member of Podocarpaceae. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 58–67.     

Ultrastructure of <Emphasis Type="Italic">Eucalyptus grandis</Emphasis> × <Emphasis Type="Italic">E. urophylla</Emphasis> plants cultivated ex vitro in greenhouse and field conditions

The young and expanded leaf micromorphology and ultrastructure of Eucalyptus grandis 2 E. urophylla juvenile plants, cultivated in greenhouse and field conditions, were analyzed by scanning and transmission electron microscopy. In greenhouse leaves epicuticular wax needles covered the abaxial and adaxial surfaces. On the adaxial surface, the needles form an atypical arrangement in lines, mainly over the anticlinal wall of epidermis cells. After plant transfer to field conditions, the organization of epicuticular wax was altered forming amorphous layers on the adaxial leaf surface, in contrast to the abaxial surface, which maintained the wax needle cover. In both culture conditions the lamellar cuticle formed on the young leaves surface disappeared during leaf enlargement. The ex vitro environment induced the development of hypostomatic leaves. The dorsiventral organization of greenhouse leaves was replaced by an isobilateral arrangement in field conditions with concomitant aerial space reduction. Results suggest that those structural changes may be some of the strategies to avoid excessive plant transpiration during Eucalyptus hybrid plants' acclimatization.     

Nanotubules on plant surfaces: chemical composition of epicuticular wax crystals on needles of Taxus baccata L

Needles of Taxus baccata L. were covered with tubular epicuticular wax crystals varying in diameters (100 and 250 nm) and lengths (300-500 and 500-1000 nm) on the abaxial and adaxial surfaces, respectively. Various sampling protocols were employed to study the chemical composition of the needle waxes on three different levels of spatial resolution. First, a dipping extraction of whole needles yielded the total cuticular wax mixture consisting of very long chain fatty acids (21%), alkanediols (19%), phenyl esters (15%), and secondary alcohols (9%) together with small amounts of aldehydes, primary alcohols, alkanes, alkyl esters, and tocopherols. Second, waxes from both sides of the needle were sampled separately by brushing with CHCl3-soaked fabric glass. Both sides showed very similar qualitative composition, but differed drastically in quantitative aspects, with nonacosan-10-ol (18%) and alkanediols (33%) dominating the abaxial and adaxial waxes, respectively. Third, the epi- and intracuticular wax layers were selectively sampled by a combination of mechanical wax removal and brushing extraction. This provided direct evidence that the tubular wax crystals contained high percentages of nonacosane-4,10-diol and nonacosane-5,10-diol on the abaxial surface, and nonacosan-10-ol on the adaxial surface of the needles. Together with these compounds, relatively large amounts of fatty acids and smaller percentages of aldehydes, primary alcohols, alkyl esters, and alkanes co-crystallized in the epicuticular layer. In comparison, the intracuticular wax consisted of higher portions of cyclic constituents and aliphatics with relatively high polarity. The formation of the tubular crystals is discussed as a spontaneous physico-chemical process, involving the establishment of gradients between the epi- and intracuticular wax layers and local phase separation.     

THE TRANSMITTING TRACT IN GLADIOLUS. I. THE STIGMA AND THE POLLEN-STIGMA INTERACTION

The stigma papillae in Gladiolus are of the “dry” type and are highly vacuolated cells with an organelle-rich peripheral cytoplasm. The cell wall of each papilla is overlain by a distinctive cuticle possessing an irregularly scalloped inner margin. Between the cell wall and cuticle is a layer of amorphous sub-cuticular material. Lipids are detected on the papilla surface. A pollen grain will hydrate and germinate only on a papilla and not on any other (non-papillate) portion of the stigma. The pollen tube penetrates the papilla cuticle, which is forced away from the papilla cell wall by sub-cuticular pollen tube growth. As the cuticle lifts away, the sub-cuticular material disperses. At the base of the papilla, the pollen tube grows onto the adaxial non-papillate surface of the stigma lobe. At this site, the cuticle has been lifted away from the underlying cells by release of a mucilaginous substance from the latter, and the pollen tube grows within this substance beneath the detached cuticle. The cytological features of Gladiolus papillae are compared with other stigma papillae described in the literature. Also, a review of the literature, as well as some of the findings of the present study, suggest that certain prevalent interpretations of dry stigma structure and function may be open to question.     

葛(豆科)叶的解剖学研究

利用光学显微镜和扫描电镜观察了葛(Pueraria lobata)叶的解剖学特征。结果表明,葛叶片的上、下表皮都只有一层表皮细胞,上表皮比下表皮厚。上、下表皮都有腺毛和非腺毛。气孔主要分布在下表皮,下表皮的气孔密度为(261±17)mm-2,上表皮只有(6±3)mm-2。叶肉由两层栅栏组织细胞和一层海绵组织细胞构成。叶肉细胞中有丰富的叶绿体。在栅栏组织和海绵组织之间有一层平行于叶脉的薄壁细胞。叶脉中含有大量的草酸钙晶体。葛叶的这些形态特征与其喜阳、耐旱的特点相适应。     

大豆叶片结构对CO_2浓度升高的反应(英)

应用光学显微镜和扫描电镜研究了ＣＯ２ 浓度对大豆（Ｇｌｙｃｉｎｅ ｍ ａｘ）叶片形态和解剖特征的影响。结果表明,叶片外部形态没有显著变化,而叶片气孔密度随ＣＯ２ 浓度升高呈下降趋势。对照组叶片上下表面和处理组的上表面均无表面角质蜡层,而处理组的下表面覆盖有大量星状的表面角质蜡层,它们在气孔区和非气孔区的数量基本差不多。此外,还发现叶肉中增加了一层栅栏组织,从而使叶片明显增厚。结果证实,ＣＯ２ 浓度增加将促进细胞分裂和表面角质蜡层的产生     

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.     

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.     

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.     

新疆5种丛藓科植物的解剖学研究

运用石蜡切片和电镜扫描技术,对5种丛藓科植物的茎、叶进行了解剖学研究。结果表明:短叶对齿藓Didymodon tectorus(C.Müll.)Saito.整个茎细胞的细胞壁均加厚,叶细胞壁凹陷呈网状,角质层纹饰条状、鳞片状和颗粒状;卷叶丛本藓Anoectangium thomsonii Mitt.茎表皮及中轴细胞壁厚,叶表面的粗疣粗糙、密集,疣上还有小孔和鳞片状纹饰;山赤藓Syntrichia ruralis(Hedw.)Web.Mohr.茎皮部细胞质浓,叶片背腹两面均密被鹿角状疣,疣上有层层叠加状纹饰和乳突;无疣墙藓Tortula mucronifolia Schweagr.茎横切面呈长圆形,叶细胞壁薄,无疣,叶背面细胞表皮较粗糙,叶腹面细胞表皮较光滑,角质层纹饰稀疏且短;长叶纽藓Tortella tortuosa(Hedw.)Limpr.茎表皮细胞壁强烈凹陷,叶细胞表面密被粗疣,疣上的角质层纹饰呈不规则鳞片状。丛藓科植物茎及叶的微观特征,为该科植物属、种的鉴定提供了重要的分类依据。     

Composition of the epicuticular and intracuticular wax layers on Kalanchoe daigremontiana (Hamet et Perr. de la Bathie) leaves

Epicuticular and intracuticular waxes from both adaxial and abaxial surfaces of the leaves of Kalanchoe daigremontiana were analyzed. All wax mixtures were found to contain approximately equal amounts of triterpenoids and very long chain fatty acid (VLCFA) derivatives. The triterpenoid fraction consisted of glutinol (8-19% of the total wax) and friedelin (4-9%), together with smaller amounts of glutanol, glutinol acetate, epifriedelanol, germanicol and β-amyrin. The VLCFA derivatives comprised C₂₇-C₃₅ alkanes (19-37% of the total wax), C₃₂-C₃₄ aldehydes (3-7%), C₃₂ and C₃₄ fatty acids (0.2-3%), C₂₆-C₃₆ primary alcohols (4-8%), and C₄₂-C₅₂ alkyl esters (2-9%). The wax layers were found to differ in triterpenoid amounts, with the intracuticular wax containing higher percentages of most triterpenoids than the epicuticular wax. Friedelin, the only triterpenoid ketone present, showed the opposite distribution with higher proportions in the epicuticular wax. VLCFA derivatives also accumulated to higher percentages in the epicuticular than in the intracuticular wax layer. Epicuticular wax crystals were observed on both the adaxial and abaxial leaf surfaces.     

Leaf cuticle ultrastructure of Pseudofrenelopsis dalatzensis (Chow et Tsao) Cao ex Zhou (Cheirolepidiaceae) from the Lower Cretaceous Dalazi Formation of Jilin,China

The cheirolepidiaceous conifer Pseudofrenelopsis dalatzensis from the Lower Cretaceous Dalazi Formation, Jilin Province, Northeast China is studied in detail based on abundant material. The gross morphology of vegetative shoots shows a greater variation than previously known. Additional information of microstructure is that the adaxial cuticle from the free part of leaves is also papillate as the abaxial cuticle. The excellent preservation of cuticles allows for the first time in this family to four distinct types of cuticle in ultrastructural level: subsidiary cells, guard cells, epidermal cells with and without papillae. Based on 30 measurements the statistic approach on each type of cell cuticle, i.e. not only the total thickness of the cuticle but also details and proportions of all different layers. A key is provided using a combination of 7 significant ultrastructural characters. These results seem to be very promising for the identity of each taxon within the Cheirolepidiaceae family. The cuticular ultrastructure of this family is characterized by having a cuticle proper A composed of A1 wavy polylamellate layer and A2 granular layer, above the cuticular layer B with B1 fibrillate and B2 granular layers. The importance of ultrastructural characters at different levels of taxonomy and palaeoecology is also discussed.     

Attachment force of the beetle Cryptolaemus montrouzieri (Coleoptera, Coccinellidae) on leaflet surfaces of mutants of the pea Pisum sativum (Fabaceae) with regular and reduced wax coverage

This study represents an investigation of surface-related plant–insect interactions. Surface micro-morphology of leaflets in pea (Pisum sativum) with wild-type crystalline surface waxes (waxy) and with reduced crystalline surface waxes (glossy) caused by a mutation (wel) were studied using various microscopy techniques. The free surface energy of these plant surfaces was estimated using contact angles of droplets of three different liquids. The morphological study of the attachment system in the ladybird beetle Cryptolaemus montrouzieri was combined with measurements of attachment (traction) forces, generated by beetles on these plant substrates. Differences were found in wax crystal shape, dimensions, and density between the adaxial and abaxial surfaces of waxy and glossy plants. The crystalline wax was not completely eliminated in the glossy plant: it was only slightly reduced on the adaxial side and underwent greater changes on the abaxial side. The free surface energy for both surfaces of both pea types was rather low with strongly predominating dispersion component. Insects generated low traction forces on all intact plant surfaces studied, except the abaxial surface of the glossy plant, on which the force was greater. After being treated with chloroform, all the surfaces allowed much higher traction forces. It is demonstrated that the difference in the crystal length and density of the epicuticular wax coverage within the observed range did not influence wettability of surfaces, but affected insect attachment. The reduction in insect attachment force on plant surfaces, covered with the crystalline wax, is explained by the decrease of the real contact area between setal tips of beetles and the substrate. Handling editor: Lars Chittka.     

Leaf surface characteristics and wetting in Ceratonia siliqua L.

Compound leaves of Ceratonia siliqua L. (carob tree) exhibit a long life span and are exposed to environmental stimuli for approximately twenty months. The micromorphology of the adaxial and the abaxial leaflet surfaces was studied, in comparison with treated waxless epidermises (after the removal of cuticle and epicuticular waxes) and corresponding replicas, respectively. The microstructural surface features are evaluated as possible consistent parameters related to the wetness of leaves. The abaxial leaflet surface is more hydrophobic than the adaxial leaflet surface in C. siliqua, which may be particularly important for the ecophysiological status of its hypostomatic leaves.     

Variability in epidermal characters of <Emphasis Type="Italic">Ginkgo tzagajanica</Emphasis> Samylina (Ginkgoales) from the Paleocene of the Tsagayan formation (Amur Region) and the taxonomy of tertiary species of <Emphasis Type="Italic">Ginkgo</Emphasis>

The study of new material on Ginkgo tzagajanica Samylina and earlier published data has shown that the epidermal morphology of the species in highly variable; several new characters were found. Occurrence of different states of characters was analyzed, their frequency distribution was revealed, and typical and rare states of characters within variation rows were determined. The specific diagnosis of G. tzagajanica was emended. The most variable characters are degree of undulation of the anticlinal walls of cells of abaxial and adaxial epidermises (the latter is more variable) and degree of development and number of papillae on ordinary epidermal cells. Less variable characters are degree of development of papillae on subsidiary cells of stomata and how the papillae cover the stomatal apertures. These are characters that bear maximal diagnostic significance for Cenozoic Ginkgo.     

Ontogenetic variations in the composition of peach leaf wax

The composition of the epicuticular waxes from the adaxial and abaxial surfaces of peach leaves varies considerably during one season's growth. Triterpenoid acids are major components 84–95% of the waxes from the youngest leaves but the proportions of these constituents decrease as the leaves expand. The waxes from the abaxial surfaces of fully expanded leaves consist primarily of hydrocarbons (C_22–C34) and triterpenoid acids, whereas the adaxial surface waxes also contain large proportions of primary alcohols (C₂₆-C₃₄) and esters (C₄₂-C₅₂). The latter include sitosteryl esters of hexacosanoic, octacosanoic and eicosanoic acids. Variations were also noted between fully expanded leaves of different ages, the abaxial surface waxes of the oldest leaves containing the highest proportions of hydrocarbons, whilst the wax from the adaxial surface of the corresponding leaves contained the largest amounts of esters, sitosterol and hydrocarbons.