Intracuticular Wax Research Articles

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 C 19 to C 27, with a prevalence of C 21, C 23 and C 25. 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.

Uptake and Storage of PCBs by Plant Cuticles

The uptake kinetics and storage of PCBs by isolated cuticles and cuticular waxes from Hedera helix, Prunus laurocerasus, and Ilex aquifolium were studied. Small chambers were used, allowing variation in plant uptake parameters to be studied by having the same air boundary layer in each chamber. During the 64 day study tri- and tetrachlorinated biphenyls generally reached equilibrium in waxes but not in whole cuticles. Differences between species were observed. Higher chlorinated PCB congeners did not approach equilibrium in either sample type. Although PCBs showed higher affinity for waxes than whole cuticles, the latter dominated the total uptake capacity on a surface area basis, because of the large amount of nonwax cuticular components. Mass transfer coefficients (MTCs) for PCB uptake (into both cuticles and waxes) indicated partition dependence up to log octanol/air partition coefficients (K(OA)) of 8.5-10, depending on species and sample type. For cuticles, higher MTCs occurred at the beginning of the experiment than later. This was not seen in reconstituted waxes, a difference which may be explained by the dispersion of intracuticular waxes within cuticles. For more lipophilic compounds, uptake appeared to be limited by diffusion processes, which may be influenced by plant physiology. Leaf surface area is, therefore, likely to control the ability of vegetation to scavenge these compounds from the air in many field situations.

Chemical Composition of the Epicuticular and Intracuticular Wax Layers on Adaxial Sides of Rosa canina Leaves

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. 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. 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. 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.

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.

Chemical composition of epicuticular wax crystals on the slippery zone in pitchers of five Nepenthes species and hybrids

Plants of the carnivorous genus Nepenthes efficiently trap insects in leaf pitchers, mostly employing epicuticular wax crystals on the pitcher walls to make them slippery for the prey. In the present study, the compositions and micromorphologies of the wax crystals of five Nepenthes species and hybrids were analysed in order to test whether the chemical principles underlying this ecological function are widespread within the genus. Three wax layers could be distinguished within the Nepenthes pitcher cuticles: (1) the outermost part of the crystals forming the platelets visible in standard scanning electron microscopy, (2) the bottom portion of the epicuticular wax crystals, and (3) an intracuticular wax layer. The composition of the intracuticular wax differed significantly from that of the neighbouring epicuticular layer. The compositions of corresponding wax mixtures from all five Nepenthes species and hybrids were very similar, with almost equal amounts of very long chain aldehydes and primary alcohols. While triacontanal (C(30) aldehyde) was prevailing in the epicuticular crystals of Nepenthes albomarginata and Nepenthes x intermedia, Nepenthes x superba and Nepenthes x henriana were found to have especially high percentages of dotriacontanal (C(32) aldehyde). Nepenthes "khasiana" had an intermediate aldehyde composition with almost equal amounts of both chain lengths.

Water transport in plant cuticles: an update

The scale, mechanism, and physiological importance of cuticular transpiration were last reviewed in this journal 5 and 10 years ago. Progress in our basic understanding of the underlying processes and their physiological and structural determinants has remained frustratingly slow ever since. There have been major advances in the quantification of cuticular water permeability of stomata-bearing leaf and fruit surfaces and its dependence on leaf temperature in astomatous surfaces, as well as in our understanding of the respective roles of epicuticular and intracuticular waxes and molecular-scale aqueous pores in its physical control. However, understanding the properties that determine the thousand-fold differences between permeabilities of different cuticles remains a huge challenge. Molecular biology offers unique opportunities to elucidate the relationships between cuticular permeability and structure and chemical composition of cuticles, provided care is taken to quantify the effects of genetic manipulation on cuticular permeability by reliable experimental approaches.

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 CHCl 3-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.

Surface Composition of Myrmecophilic Plants: Cuticular Wax and Glandular Trichomes on Leaves of Macaranga tanarius

Primary plant surfaces, covered with cuticles consisting of cutin and waxes, are important substrates for interaction with insects. The composition of leaf surfaces of the myrmecophilic plant Macaranga tanarius was studied. The prenylated flavanone nymphaeol-C was identified in surface extracts and was localized exclusively in glandular trichomes on the abaxial leaf side. The epidermal pavement cells surrounding these trichomes were covered with a smooth film of epicuticular wax from which few small wax crystals protruded. The epicuticular wax amounted to approximately 8 microg cm(-2), corresponding to 85% of the wax load on the adaxial as well as the abaxial leaf sides. The epicuticular wax mixtures from both leaf surfaces contained more than 70% primary alcohols, 14% fatty acids, 2% aldehydes, and traces of alkyl acetates, with chain lengths ranging from C(20) to C(38). In contrast, the intracuticular wax layer was largely dominated by triterpenoid alcohols alpha-amyrin, beta-amyrin, and lupeol. Consequently, these characteristic compounds are not available for direct contact with insects on the plant surface.

The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis.

The interface between plants and the environment plays a dual role as a protective barrier as well as a medium for the exchange of gases, water, and nutrients. The primary aerial plant surfaces are covered by a cuticle, acting as the essential permeability barrier toward the atmosphere. It is a heterogeneous layer composed mainly of lipids, namely cutin and intracuticular wax with epicuticular waxes deposited on the surface. We identified an Arabidopsis thaliana activation tag gain-of-function mutant shine (shn) that displayed a brilliant, shiny green leaf surface with increased cuticular wax compared with the leaves of wild-type plants. The gene responsible for the phenotype encodes one member of a clade of three proteins of undisclosed function, belonging to the plant-specific family of AP2/EREBP transcription factors. Overexpression of all three SHN clade genes conferred a phenotype similar to that of the original shn mutant. Biochemically, such plants were altered in wax composition (very long fatty acid derivatives). Total cuticular wax levels were increased sixfold in shn compared with the wild type, mainly because of a ninefold increase in alkanes that comprised approximately half of the total waxes in the mutant. Chlorophyll leaching assays and fresh weight loss experiments indicated that overexpression of the SHN genes increased cuticle permeability, probably because of changes in its ultrastructure. Likewise, SHN gene overexpression altered leaf and petal epidermal cell structure, trichome number, and branching as well as the stomatal index. Interestingly, SHN overexpressors displayed significant drought tolerance and recovery, probably related to the reduced stomatal density. Expression analysis using promoter-beta-glucuronidase fusions of the SHN genes provides evidence for the role of the SHN clade in plant protective layers, such as those formed during abscission, dehiscence, wounding, tissue strengthening, and the cuticle. We propose that these diverse functions are mediated by regulating metabolism of lipid and/or cell wall components.

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.

Cuticular waxes of Arabidopsis.

Cuticular waxes of Arabidopsis.

Effect of Temperature on Enzymatic and Physiological Factors Related to Chilling Injury in Carambola Fruit (Averrhoa carambola L.)

Three groups of carambola fruits (Averrhoa carambola L.) were stored at 2 and 10°C (85–90% relative humidity). The major physicochemical, physiological, and enzymatic responses of fruit were measured in each group over a 30-day period: chilling injury index (CII), decay (%), intracuticular waxes, cuticle permeability, pulp firmness, weight loss, sucrose, fructose and glucose contents, ion electrolyte leakage in pulp (%), ethylene and carbon dioxide production rates, and the activities of peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) enzymes. CII values were statistically different at 2 and 10°C, showing high significance with respect to sucrose content and weight loss (P < 0.05). Chilling injury included darkened ribs and skin desiccation. According to the CI symptom development, a possible relationship of POD and PPO activities was found at 2°C. A significant sucrose content increase was observed at 10°C. CI symptoms were associated with POD and PAL activities.

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.

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

ABSTRACTThe 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.

Structure and dynamics of reconstituted cuticular waxes of grape berry cuticle (Vitis vinifera L.)

within the matrix, intracuticular waxes or deposited on the outer surface of the plant cuticle, epicuticular waxes. Cuticular waxes from grape berry cuticle of Vitis vini- Cuticular waxes constitute the main barrier limiting the fera L. have been investigated by X-ray diffraction, transport across the plant-atmosphere interface. This prop- differential scanning calorimetry and gas chromato- erty allows waxes to control cuticular transpiration graphy. The waxes were mainly composed of n-alco- (Schonherr, 1982), foliar uptake of xenobiotics (Schonherr hols and n-fatty acids and a considerable amount and Riederer, 1989; Schreiber and Schonherr, 1993) and (about 30%) of the cyclic terpenoid oleanolic acid. The resistance against fungi (Commenil et al., 1997). This is due physical techniques used showed that the waxes have to their partial arrangement in crystalline regions, where a high degree of molecular order in spite of the pres- the middle portions of the long aliphatic chains of the wax ence of the cyclic component. Molecular dynamics of constituents are regularly aligned, and in amorphous the reconstituted waxes were measured to character- regions where short-chain aliphatics and cyclic compounds ize the transport properties of the cuticular waxes that form clusters outside crystalline regions (Riederer and form the transport-limiting barrier of plant cuticles. Schreiber, 1995). Those waxes can, in fact, form crystalline For this purpose, the diffusion coefficient of labelled platelets oriented parallel to the surface of the cuticular cholesterol, imitating the terpenoic acid, was meas- membrane and these have been demonstrated using polar- ured. The value obtained was around 10'21 m2 s'1 ized light (Sitte and Rennier, 1963). indicating a low mobility of the cyclic part of the recon- Permeability of cuticles diVers greatly among plant stituted waxes. Temperature dependence of the dif- species and it has been argued that both the chemistry fusion coefficient was studied in the range of 5-45°C. and the structure of cuticular waxes are responsible for Arrhenius plot analysis yielded a high activation these diVerences (Riederer and Schonherr, 1985). Most energy, 196.4 kJ mol'1, of the diffusion process. This studies have been carried out in plant species where indicates dense molecular packing of reconstituted epicuticular waxes are mainly composed of long aliphatic cuticular waxes. molecular chains. In this sense, current knowledge of the fine structure and dynamics of diVerent plant cuticular

Cires epi- et intracuticulaires: Etude de leur separation, de leurs caracteristiques chimiques et de leurs rôles respectifs dans la permeabilité cuticulaire

The use of a cellulose nitrate film (with acetone 6% weight/volume) facilitated the stripping and recovery of the epi- and intracuticular waxes from the two surfaces of Eucalyptus leaves. This study showed that depending on the species, the epicuticular wax ( E. globulus) or the intracuticular wax ( E. gunnii) may be the predominant wax of the leaf. Generally speaking, the quantity of intracuticular wax is independent of the surface, but the epicuticular wax is always more abundant on the abaxial surface. The leaf aging process reduces the difference between the surfaces, and induces a modification of the wax chemical composition. The alkane component has a shorter chain length (disappearance of C 30 and C 31, and appearance of C 24 and C 26), with the presence of alkenes only in the mature leaves. When an inhibitor of wax biosynthesis, e.g. trichloroacetic acid, is supplied in the culture solution, the epicuticular wax shows a large decrease in the polar fraction; and correlates inversely with a decrease of βΘ-dicetone in the apolar fraction, only a decrease of the long chain alkanes (C 29 and C 31) correlates with an increase of the shorter chains (C 25 and C 27). In the intracuticular wax, the apolar fraction decreases, but with few modifications in the proportions of the different alkanes. This physiological effect of TCA, essentially observable at the level of the chemical composition of the epicuticular wax, confirms that epi- and intracuticular wax biosynthesis pathways are different. Our technique of stripping permits us to study the water resistance of the different components of isolated cuticle of Prunus laurocerasus and to show that at the wax level, the intracuticular wax fraction is the main shield against water flow with a complementary role of the cutin matrix.

Structural changes and permeability of ivy (Hedera helix L.) leaf cuticles in relation to leaf development and after selective chemical treatments

summaryThe fine structure of ivy leaf cuticles was investigated as a function of leaf development before and after cuticle isolation as well as after selective extraction of cuticle components. Cuticle mass increased with increasing age from 234°3 to 539.1 μg cm−2 Waxes increased from 12.3 to 18.6%, of cuticle mass from young to old leaves. However, percentages of cutin and non‐lipid constituents did not vary significantly with leafage. They represented approx. 58 and 26%, of the cuticle mass, respectively. Cuticle thickness determined from electron and light microscopy increased 12–fold during leaf growth to reach 4.25 μm for mature leaves. Transmission electron microscopy of transverse sections of non‐isolated and isolated cuticles showed an outer lamellate zone gradually merging from an inner reticulate zone the thickness of which increased with leaf growth. The lamellate zone appeared very early (in vitro unexpanded leaf) and was characterized by a constant thickness (0.2 μm). Cuticle lamellation seemed to disappear after extraction of soluble cuticular wax, which confirmed intracuticular wax localization in lamellae. Electron‐dense fibrillae observed in the reticulate zone disappeared after acid hydrolysis showing that they are made of non‐lipid components. X‐ray diffraction confirmed the presence of crystalline structures and revealed a cuticle disorganization alter wax extraction. Permeability measurements showed an increase rather than a decrease in glyphosate and isoproturon diffusion through cuticles with increasing leafage, suggesting that the main barrier to diffusion is the outer lamellate zone. A considerable increase in isoproturon penetration through cuticles resulted from the wax extraction, demonstrating the major role of wax in the control of cuticular permeability to this herbicide.

Discrimination between epicuticular and intracuticular wax in blackberry leaves: Ultrastructural and chemical evidence

Epicuticular wax on the adaxial leaflet side of blackberry ( Rubus fruticosus) occurred as wax knobs and irregular-shaped platelets. Removal of the epicuticular wax by a collodion film revealed the wax-free cuticle surface. This allowed the selective extraction of epicuticular wax from the film and subsequently of intracuticular wax from the cuticle. Epicuticular wax amounted to approx. 90% of total cuticular wax, only about 10% were present as intracuticular wax. The epicuticular wax contained mainly alcohol acetates, free alcohols and esters with lesser amounts of fatty acids and alkanes. Intracuticular wax was composed of free alcohols, alcohol acetates and fatty acids together with small proportions of triterpenoid acids, alkanes and esters.

Ultrastructure and Chemistry of Cuticular Waxes of Developing Citrus Leaves and Fruits1

Abstract The ultrastructure of leaf and fruit surface waxes of citrus developed similarly. The wax layers of immature leaves and fruits were initially amorphous. Small protrusions and isolated regions of upright platelets then developed. All surfaces eventually cracked and uplifted to form large flat irregular plates. Epicuticular wax concentrations (μg/cm2) of new spring flush leaves were initially high, declined rapidly in March-April, and increased to June maxima after which levels declined slightly. Fruit epicuticular wax concentration generally declined in May-June and then increased into November. Secondary alcohols dominated citrus leaf waxes in March-April but quickly decreased or were diluted to trace levels. Overall, the major citrus leaf wax fractions were primary alcohols, with paraffins 2nd in importance. Aldehydes and fatty acids were dominant in citrus fruit waxes. For each cultivar, the relative proportions of individual wax fractions changed during the year. The intracuticular waxes were mainly fatty acids. The relationships between wax ultrastructure and chemistry in developing leaves and fruits are discussed.

Of the adjustment of a quadrilateral in a tertiary system of triangulation

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.