In vitro Reconstitution of Epicuticular Wax Crystals: Formation of Tubular Aggregates by Long-Chain Secondary Alkanediols

Cuticles of several plant species are covered by tubular wax aggregates that are known to consist mainly of (S)-nonacosan-10-ol. The present work addresses the question whether minor wax components may additionally contribute to these tubules. Thin layer chromatography was used to prepare secondary alkanediol fractions from leaf cuticular waxes of Nelumbo nucifera and Thalictrum flavum, containing nonacosane-3,10-diol, nonacosane-4,10-diol, nonacosane-5,10-diol, nonacosane-7,10-diol, nonacosane-9,10-diol and nonacosane-10,13-diol. From organic solutions all these compounds crystallized in tubular shapes. Possible crystal structures of relevant alkanediol isomers are proposed, in analogy to the lattice geometries of comparable aliphatic compounds. The resulting structural model shows that nonacosan-10-ol and various secondary alkanediols may join in metastable mixed crystals. According to the structural model proposed the admixture of alkanediols to nonacosan-10-ol aggregates should enhance the stability of their tubular habit.     

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 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.     

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.     

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.     

Very long chain alkylresorcinols accumulate in the intracuticular wax of rye (Secale cereale L.) leaves near the tissue surface

Alkylresorcinols (ARs) are bioactive compounds occurring in many members of the Poaceae, likely at or near the surface of various organs. Here, we investigated AR localization within the cuticular wax layers of rye (Secale cereale) leaves. The total wax mixture from both sides of the leaves was found to contain primary alcohols (71%), alkyl esters (11%), aldehydes (5%), and small amounts (<3%) of alkanes, steroids, secondary alcohols, fatty acids and unknowns. A homologous series of ARs (3%) was identified by GC-MS and comparison with a synthetic standard of nonadecylresorcinol. The alkyl side chains of the wax ARs contained odd numbers of carbons ranging from C19 to C27, with a prevalence of C21, C23 and C25. Waxes from both sides of the leaf, analyzed separately in a second experiment, comprised the same compound classes in similar relative amounts and with similar homolog patterns. Finally, the epicuticular and intracuticular wax layers were sampled separately from the abaxial side of the leaf. While ARs accounted for 2% of the intracuticular wax, they were not detectable in the epicuticular wax. The intracuticular wax was also slightly enriched in steroids, whereas the epicuticular layer contained more primary alcohols. All other wax constituents were distributed evenly between both wax layers.     

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.     

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.     

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.     

Epicuticular wax of Pinus radiata needles

Epicuticular wax isolated from the cotyledons and primary needles of 10-week-old Pinus radiata seedlings is similar in composition and contains 86% neutral compounds, viz. alkyl esters (25%, C₂₄–C₆₄), nonacosan-10-ol (52%), heptacosane-5,10-diol (2%), nonacosane-4,10-diol, nonacosane-5,10-diol, and nonacosane-10,13-diol (total 12%) and estolides, MW ca 800 (2%), MW ca 1100 (6%), and MW ca 1500 (1%). The acidic fraction (14%) contains n-acids (78%, C₁₂–C₃₂) and diterpene acids (22%, mainly abieta-8,11,13-trien-18-oic, with lesser amounts of pimara-8(14),15-dien-18-oic, isopimara-7,15-dien-18-oic and hydroxylated aromatic, diene and mono-ene acids). Wax isolated from primary needles of 1-yr-old seedlings had a similar neutral fraction composition, but the acidic fraction contained predominantly the diterpene acid mixture, with only trace amounts of n-acids. The wax from 1-yr-old secondary, needles from P. radiata forest trees aged 5 yr and 40 yr contained an acid fraction (12% 5 yr, 17% 40 yr trees) comprising the diterpene acid mixture, with trace amounts of n-acids together with ω-hydroxy acids (C₁₂, C₁₄ and C₁₆). The neutral fraction from both young and old trees had a similar composition containing alkyl esters (7%, C₂₄–C₆₆), estolides (90%, MW 566-ca 1500), nonacosan-10-ol (2%) and the heptacosane and nonacosane diols (1%). During growth and maturation of P. radiata, the nonacosan-10-ol content of the needle wax decreases while the proportion of estolides and diterpene acids increases, the latter probably being located around the stomatal pore.     

Epicuticular wax of Juniperus scopulorum

Epicuticular wax from Juniperus scopulorum contains hydrocarbons (16%), esters (11%), free acids (1%), nonacosan-10-ol (27%), nonacosane-diols (7%) and estolides (16%). The major hydrocarbon is tritriacontane; the principal esters are C₃₄–C₄₆, mainly octyl and decyl esters of C₂₈-C₃₆ acids; and the diols consist of nonacosane-4,10-diol (57%),5,10-diol (28%), 7,10-diol (11%) and 10,13-diol (4%). Hydrolysis of the estolides gave a mixture of acids, ω-hydroxy acids, , ω-diols, alcohols and hydroxy acids. The hydroxy acids are a new class of C₂₈–C₃₆ acids with the OH attached to either the eighteenth or twentieth carbon from the terminal methyl end; the major component is 13-hydroxydotriacontanoic acid. Syntheses of this acid and of nonacosane-4,10-dione and nonacosane-4,10-diol are described.     

Identification of the diol associated with variations in wax ultrastructure on Rhus cotinus leaves

The diol constituent of Rhus cotinus leaf epicuticular wax has been identified as nonacosane-5,10-diol from chemical investigations of the free compound, the TMSi ether and the nonacosane-5,10-dione prepared from the diol by oxidation. The form and distribution of the crystalline waxes changed as the leaves expanded, dense clusters of short tubes covering the thin ribbons formed during the initial stages of growth. The diol content of the wax decreased by more than 50% over the same period.     

Studies on the structure of the plant wax nonacosan-10-ol, the main component of epicuticular wax conifers

The main component presents in the epicuticular waxes of needles of Pinus halepensis and the most of conifers, the secondary alcohol nonacosan-10-ol, has been investigated by X-ray diffraction and differential scanning calorimetry. The results obtained from these physical techniques permitted to establish a definitive structural model of the molecular arrangement of this wax, basically in good agreement with the model formulated by other authors from theoretical formulations. Biological implications of the proposed structure have been also formulated.     

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.     

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.     

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.     

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.     

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.     

Changes of epicuticular wax induced by enhanced UV-B radiation impact on gas exchange in Brassica napus

The epicuticular wax covering on plant surface plays important roles in protecting plants against UV radiation. However, the role of epicuticular wax in affecting leaf gas exchange under enhanced ultraviolet-B (UV-B) radiation remains obscure. In the present study, different aged leaves of Brassica napus were used to analyze the responses of crystal structure and chemical constituents of epicuticular wax to UV-B radiation and the effects of such responses on gas exchange indices. Enhanced UV-B radiation significantly decreased the amount of esters in all leaves except the first leaf, amount of secondary alcohols in the second, third and fourth leaves, and amount of primary alcohols in the second and third leaves, while increased the amounts of ketones and aldehydes in the first leaf. Enhanced UV-B level had no significant effect on the amounts of alkanes and total wax in all leaves. Exposure to UV-B radiation resulted in wax fusion on adaxial leaf and stomata opening on abaxial leaf. Fusions of plates and rods on adaxial leaf surface covered most of the stomata, thereby influencing the photosynthesis in the upper mesophyll of leaves. Enhanced UV-B level significantly reduced the net photosynthesis rate (P _N) but increased the stomata conductance (g _s), concentrations of intercellular CO₂ (C _i ), and transpiration rate (E) in all leaves. Both UV-B radiation and the wax fusion induced by enhanced UV-B radiation resulted in different stomata status on abaxial and adaxial leaf surface, causing decrease of P _N, and increase of g _s, C _i and E in leaves.