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.     

Effect of alterations in cuticular wax biosynthesis on the physicochemical properties and topography of maize leaf surfaces

The leaf surface properties of 11 cuticular wax mutants of maize were characterized, and this information was used to identify the quantitative relations among distinct leaf surface traits. Compared with the wild‐type maize, these mutants were reduced 3–24% in their leaf surface hydrophobicity, 20–88% in the mass of cuticular waxes on their leaves, and 52–94% in the percentage of planar leaf surface area covered with epicuticular crystalline waxes. They also differed in the presence and abundance of the epicuticular crystalline waxes in each of seven structural classes. With the exception of one mutant, the mass of cuticular waxes produced by these mutants was positively correlated with the number of epicuticular crystalline waxes per unit area on their leaves. Furthermore, an increase of 0·4 mg of cuticular wax per gram of leaf (dry weight) was associated with a 1% increase in leaf surface area covered by epicuticular crystalline waxes, and this 1% increase was associated with a 2° increase in the contact angle of a water droplet on the leaf surface. Linear differences in the leaf surface hydrophobicity were associated with exponential differences in the mass of the cuticular waxes produced. Quantitative knowledge of these leaf surface properties is highly relevant to the interactions of leaves with environmental factors such as microbes, insects, agricultural chemicals, and pollutants.     

Abnormal germling development by brown rust and powdery mildew on cer barley mutants

The barley leaf rust fungus forms appressoria over host leaf stomata and penetrates via the stomatal pore. High levels of avoidance to leaf rust fungi have been described in some wild accessions of Hordeum species where a prominent wax layer on the stomata inhibits triggering of fungal appressorium differentiation. Leaf rust avoidance has not yet been found in H. vulgare. Since cuticular leaf waxes are implicated in the avoidance trait, we screened 27 eceriferum (cer) mutant lines of H. vulgare for avoidance to barley leaf rust. These mutations affect leaf waxes. Reduction in numbers of germ tubes forming appressoria over stomata was found in some lines, but the greatest reduction (ca 30%) was less than previously found in wild barley spp. or in an accession of H. chilense used here as a check. In one line (cer-zh654), avoidance was due to a combination of factors. Firstly, fewer germ tubes oriented towards stomata and so failed to contact them. Secondly, some germ tubes that encountered stomata did not form appressoria but over-grew them. In this line, therefore, the fungus tended to fail both to locate and to respond to stomata. The appressoria of barley powdery mildew form on leaf epidermal cells that they penetrate directly. On certain cer lines, a proportion of germlings of the barley powdery mildew fungus developed abnormally, suggesting that germlings failed to recognise and/or respond to the leaf surface waxes on these mutants.     

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.     

Role of Pear Fruit Cuticular Wax and Surface Hydrophobicity in Regulating the Prepenetration Phase of Alternaria alternata Infection

The role of cuticular wax and the surface hydrophobicity of the fruit of the ‘Zaosu’ pear (Pyrus bretschneideri Rehd) in regulating the prepenetration phase of Alternaria alternata infection were analysed in vivo and in vitro. Results showed that cuticular wax on an intact fruit surface, as well as wax extracts mounted on silanized glass slides or onion epidermis, favoured the formation of short, differentiated germ tubes and large numbers of appressoria (APP) or infected hyphae (IH). Dewaxed fruits or no wax extract mounted on in vitro surfaces, however, enhanced germ tube elongation and inhibited or delayed the formation of infection structure. High surface hydrophobicity resulting from cuticular wax also stimulated infection structure formation, as contact angle (hydrophobicity) was positively correlated with APP formation but negatively correlated with germ tube elongation. Alternaria alternata cutinase enzyme activity was also induced by cuticular wax, both in vivo and in vitro. These findings suggest that the chemical composition and hydrophobicity of pear fruit cuticular wax are essential in facilitating fungal invasion by regulating the growth and differentiation of A. alternata during the prepenetration phase.     

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.     

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.     

The effects of superficial wax on leaf wettability

Experiments are described which provide more information on the role played by superficial waxes in the natural water-repellency of leaf surfaces. Contact angles of water were measured on a variety of leaf surfaces, before and after removal of wax, and on smooth films of the isolated superficial waxes. The differences in wettability of leaf surfaces are not wholly accounted for by differences which occur in the chemical and hydrophobic properties of their superficial waxes. Waxes isolated from leaves exhibiting contact angles less than 90° are usually more hydrophobic than the leaf surface itself. On most leaves exhibiting angles greater than 90° wax is the dominant factor governing water-repellency, the isolated wax normally making at least a 60 % contribution to the contact angle measured on the leaf surface. Additional factors, such as roughness, responsible for the occurrence of contact angles greater than 110° on certain leaf surfaces, reside in the wax layer. The hydrophobic properties of some leaves are unaffected by chloroform washing, revealing that superficial waxes play little part in their wettability.     

The impact of epicuticular wax on gas-exchange and photoinhibition in Leucadendron lanigerum (Proteaceae)

This study investigated the seasonal modification of wax deposition, and the impact of epicuticular wax on gas-exchange as well as photoinhibition in Leucadendron lanigerum, a species from the Proteaceae family with wax-covered leaf surfaces and the stomata also partially occluded by wax. The results of this study demonstrated that the deposition of epicuticular wax in L. lanigerum is dependent on the age of the leaf as well as the season, and generation and regeneration of wax occur mostly in spring while transformation and also degeneration of wax crystals occur in winter. Epicuticular waxes decreased cuticular water loss, but had little impact on leaf reflectance. The temperature of leaves without wax was lower than that of wax-covered leaves, indicating that the rate of transpiration impacted more on leaf temperature than reflectance of light in the PAR range in L. lanigerum. The wax coverage at the entrance of stomata in L. lanigerum increased resistance to gas diffusion and as a consequence decreased stomatal conductance, transpiration and photosynthesis. Also, the results indicated that epicuticular waxes do help prevent photodamage in L. lanigerum, and so this property could benefit plants living in arid environments with high solar radiation.     

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.     

Contact Areas between Waxy Leaf Surfaces and Aqueous Solutions: Quantitative Determination of Specific Leaf Surface Contact Areas

The contact areas (on a molecular scale) between aqueous solutionsand leaf surfaces have been measured in order to obtain informationon the actual areas effective in cuticular transport. For thispurpose, monolayer adsorption of pentachlorophenol (PCP) wasused on leaf surfaces and on artificial surfaces. The adsorptionisotherms were analysed according to the Brunauer-Emmett andTeller (BET) formalism leading to BET constants and actual contactareas between the aqueous PCP solution and the surfaces. BETconstants ranged from 4 to 11, with an arithmetic mean of 6·5.No relationship was found between the chemical composition ofthe surface waxesand the BET constants, and there were no significantdifferences between BET constants measured using isolated waxesor intact leaves. This indicates that the surfaces of the waxesare made up mainly of methylene and methyl groups, while mostfunctional groups appear not to contribute significantly tosurface sorption of PCP. This conclusion is supported by theobservation that the BET constant measured on silanized glasshaving a pure CH_3–surface was 5·7, which was notsignificantly different from 6·5. Specific leaf surfacecontact areas calculated from the BET constant and the areaof an adsorbed PCP molecule ranged from 1·6 (Hordeumvulgare) to 137 (Abies koreana). This means that the areas ofcontact between aqueous solutions and surface waxes were 1·6to 137 times larger than the apparent surface areas, which mightbe the consequence of a fractal leaf surface morphology. A leafsurface contact area greater than unity can result only froma rough surface that is wetted by the solution. It is argued,that interspecific differences in the rates of cuticular permeationmay be largely due to differences in leaf surface contact areasand not to differences in cuticular permeabilities. Consequently,differences in the rates of cuticular permeation can be interpretedonly if the leaf surface contact area that is effective in transportis known. Key words: Cuticular transport, epicuticular wax, fractality, specific leaf surface contact area, plant cuticle     

Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid beta-ketoacyl-CoA synthase

Cuticular waxes play a pivotal role in limiting transpirational water loss across the plant surface. The correlation between the chemical composition of the cuticular waxes and their function as a transpiration barrier is still unclear. In the present study, intact tomato fruits (Lycopersicon esculentum) are used, due to their astomatous surface, as a novel integrative approach to investigate this composition- function relationship: wax amounts and compositions of tomato were manipulated before measuring unbiased cuticular transpiration. First, successive mechanical and extractive wax-removal steps allowed the selective modification of epi- and intracuticular wax layers. The epicuticular film consisted exclusively of very-long-chain aliphatics, while the intracuticular compartment contained large quantities of pentacyclic triterpenoids as well. Second, applying reverse genetic techniques, a loss-of-function mutation with a transposon insertion in a very-long-chain fatty acid elongase beta-ketoacyl-CoA synthase was isolated and characterized. Mutant leaf and fruit waxes were deficient in n-alkanes and aldehydes with chain lengths beyond C30, while shorter chains and branched hydrocarbons were not affected. The mutant fruit wax also showed a significant increase in intracuticular triterpenoids. Removal of the epicuticular wax layer, accounting for one-third of the total wax coverage on wild-type fruits, had only moderate effects on transpiration. By contrast, reduction of the intracuticular aliphatics in the mutant to approximately 50% caused a 4-fold increase in permeability. Hence, the main portion of the transpiration barrier is located in the intracuticular wax layer, largely determined by the aliphatic constituents, but modified by the presence of triterpenoids, whereas epicuticular aliphatics play a minor role.     

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.     

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.     

Appressorium morphogenesis and cell cycle progression are linked in the grass powdery mildew fungus Blumeria graminis

Conidial germination and differentiation – the so-called prepenetration processes – of the barley powdery mildew fungus (Blumeria graminis f. sp. hordei) are essential prerequisites for facilitating penetration of the host cuticle. Although the cell cycle is known to be pivotal to cellular differentiation in several phytopathogenic fungi there is as yet no information available concerning the relationship between cell cycle and infection structure development in the obligate biotroph B. graminis. The timing of specific developmental events with respect to nuclear division and morphogenesis was followed on artificial and host leaf surfaces by 4′,6-diamidino-2-phenylindole (DAPI) staining in combination with a pharmacological approach applying specific cell cycle inhibitors. It was found that the uninucleate conidia germinated and then underwent a single round of mitosis 5–6 h after inoculation. During primary germ tube formation the nucleus frequently migrated close to the site of primary germ tube emergence. This nuclear repositioning was distinctly promoted by very-long-chain aldehydes that are common host cuticular wax constituents known to induce conidial differentiation. The subsequent morphogenesis of the appressorial germ tube preceded mitosis that was spatially uncoupled from subsequent cytokinesis. Blocking of S-phase with hydroxyurea did not inhibit formation of the appressorial germ tube but prevented cytokinesis and appressorium maturation. Benomyl treatment that arrests the cell cycle in mitosis inhibited nuclear separation, cytokinesis, and formation of mature appressoria. Thus, we conclude that a completed mitosis is not a prerequisite for the formation and swelling of the appressorial germ tube, which normally provides the destination for one of the daughter nuclei, while appressorium maturation depends on mitosis.     

Wetting of the upper needle surface of Abies grandis: influence of pH, wax chemistry and epiphyllic microflora on contact angles

Wetting of the upper needle surface of Abies grandis Lindl. by aqueous solutions of different pH values was investigated. With increasing needle age, contact angles decreased significantly from about 75° on current-year needles to values lower than 30° on 4-year-old needles. On older needles, contact angles were significantly lower, by more than 10°, when aqueous solutions of pH9-0 were used compared with those of pH3-0. On the surfaces of older needles, contact angle titrations were carried out, contact angles being measured with aqueous solutions covering a pH range from 3.0 to 11.0. Measured titration curves showed clear inflection points around pH 7.0, indicating the existence of ionizable carboxylie groups in the interface between needle surface and atmosphere. The evidence seems convincing that the pronounced pH dependence of wetting is mainly due to the presence and/or activity of epiphyllic micro-organisms, whereas the cuticular wax composition of Abies grandis needles does not appear to contribute significantly to this phenomenon. Thus, the results presented here allow the general conclusion that changes of contact angles measured on leaf surfaces may not always be due to changes in the leaf surface chemistry and/or the fine structure of leaf surface waxes, but may also be due to increased amounts of epiphyllic micro-organisms significantly altering the leaf surface wetting properties.     

Plant–Microbe Interactions: Wetting of Ivy (Hedera helix L.) Leaf Surfaces in Relation to Colonization by Epiphytic Microorganisms

Abstract Leaf wettability, cuticular wax composition, and microbial colonization of upper and lower leaf surfaces of ivy (Hedera helix L.) was investigated for young and old leaves sampled in June and September. Contact angles of aqueous buffered solutions measured on young leaf surfaces ranged between 76° and 86° and were not dependent on the pH value of the applied droplets. Contact angles measured on old leaf surfaces were up to 32°, significantly lower than on young leaf surfaces. Furthermore, contact angles were significantly lower using aqueous solutions of pH 9.0 compared to pH 3.0, indicating the influence of ionizable functional groups on leaf surface wetting properties. Observed changes in leaf wetting properties did not correlate with different levels of alkanoic acids in cuticular waxes. However, microscopic examination of the leaf surfaces indicated the influence of epiphytic microorganisms on wetting properties of old leaves, since their surfaces were always colonized by epiphytic microorganisms (filamentous fungi, yeasts, and bacteria), whereas surfaces of young leaves were basically clean. In order to analyze the effect of epiphytic microorganisms on leaf surface wetting, surfaces of young and clean ivy leaves were artificially colonized with Pseudomonas fluorescens. This resulted in a significant increase and a pH dependence of leaf surface wetting in the same way as it was observed on old ivy leaf surfaces. From these results it can be deduced that the native wetting properties of leaf surfaces can be significantly masked by the presence of epiphytic microorganisms. The ecological implications of altered wetting properties for microorganisms using the leaf/atmosphere interface as habitat are discussed. Received: 20 March 1999; Accepted: 5 July 1999; Online Publication: 18 July 2000     

Cuticular wax load and surface wettability of leaves and fruits collected from sweet cherry (<Emphasis Type="Italic">Prunus avium</Emphasis>) trees grown under field conditions or inside a polytunnel

In this work, the effect of microclimate at the tree level inside a polytunnel and in an uncovered orchard of Prunus avium was studied through the observation of leaf and fruit micromorphology and the accumulation of cuticular waxes. Records of environmental parameters showed a significantly higher daytime temperature in the polytunnel (on an average 4°C), whereas the night temperature did not differ between treatments. Furthermore, photosynthetic active radiation and UV-B radiation inside the polytunnel were 43 and 97% lower, respectively, than the radiation measured outside. Leaves grown in the polytunnel had lower contact angles and higher load of cuticular wax per unit area, irrespective of cultivar. The impact of the microclimate was observed at the first sampling and did not increase with exposure time of leaves. Fruits of the cultivars ‘Souvenir’ and ‘Prime Giant’ grown outside had significantly less cuticular wax despite their delayed harvest time of 11 and 5 days, respectively. The relationship of higher cuticular wax load and lower contact angles of protected leaves might have practical importance for plant protection activities and the retention and uptake of leaf-applied agrochemicals.     

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.