Plant Epicuticular Waxes Research Articles

Crystallinity of plant epicuticular waxes: electron and X-ray diffraction studies

The crystal structure of the epicuticular waxes of 35 plant species has been examined by electron diffraction and X-ray powder diffraction. The waxes include the most common morphological wax types such as platelets, tubules, films and rodlets. Most of them were prepared with a special mechanical isolation method, which preserves the original crystal structure. Solvent-extracted recrystallized plant waxes were compared with mechanically isolated samples. The waxes were found to occur in three different crystal structures. Most of the waxes exhibited an orthorhombic structure which is the most common for aliphatic compounds. Tubules containing mainly secondary alcohols showed diffraction reflections of a triclinic phase; broad reflection peaks indicated a significant disorder. Ketones, in particular beta-diketone tubules, displayed the reflections of a hexagonal structure. Mixtures of different phases could be identified. For most of the waxes, the ‘long spacing’ diffraction reflections indicated a layer structure with the characteristics of the major component. Others showed no ‘long spacing’ reflections indicating a strong disorder of the molecular layers.

Structural analysis of wheat wax (Triticum aestivum, c.v. ‘Naturastar’ L.): from the molecular level to three dimensional crystals

In order to elucidate the self assembly process of plant epicuticular waxes, and the molecular arrangement within the crystals, re-crystallisation of wax platelets was studied on biological and non-biological surfaces. Wax platelets were extracted from the leaf blades of wheat (Triticum aestivum L., c.v. 'Naturastar', Poaceae). Waxes were analysed by gas chromatography (GC) and mass spectrometry (MS). Octacosan-1-ol was found to be the most abundant chemical component of the wax mixture (66 m%) and also the determining compound for the shape of the wax platelets. The electron diffraction pattern showed that both the wax mixture and pure octacosan-1-ol are crystalline. The re-crystallisation of the natural wax mixture and the pure octacosan-1-ol were studied by scanning tunnelling microscopy (STM), atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Crystallisation of wheat waxes and pure octacosano-1-ol on the non polar highly ordered pyrolytic graphite (HOPG) led to the formation of platelet structures similar to those found on the plant surface. In contrast, irregular wax morphologies and flat lying plates were formed on glass, silicon, salt crystals (NaCl) and mica surfaces. Movement of wheat wax through isolated Convallaria majalis cuticles led to typical wax platelets of wheat, arranged in the complex patterns typical for C. majalis. STM of pure octacosan-1-ol monolayers on HOPG showed that the arrangement of the molecules strictly followed the hexagonal structure of the substrate crystal. Re-crystallisation of wheat waxes on non-polar crystalline HOPG substrate showed that technical surfaces could be used to generate microstructured, biomimetic surfaces. AFM and SEM studies proved that a template effect of the substrate determined the orientation of the re-grown crystals. These effects of the structure and polarity of the substrate on the morphology of the epicuticular waxes are relevant for understanding interactions between biological as well as technical surfaces and waxes.

Policosanol Contents and Compositions of Wheat Varieties

Policosanol (PC) is the common name for a mixture of high molecular weight (20-36 carbon) aliphatic primary alcohols, which are constituents of plant epicuticular waxes. Wheat germ oil has been reported to improve human physical fitness, and this effect is attributed to its high PC, specifically its high octacosanol (OC) content. Although the PC composition of wheat leaves has been studied extensively, information on PC content and composition of wheat grain fractions is scarce. The objective of this study was to examine the PC contents and compositions of wheat grain fractions of 31 varieties grown in Oklahoma. PC compositions of the samples were identified using a gas chromatograph coupled with a mass spectrometer. The PC content of wheat bran was higher than that of the germ, shorts, and flour. The Trego and Intrada varieties had the highest PC content among the 31 wheat varieties studied. Tetracosanol (C24), hexacosanol (C26), and OC (C28) were the major PC components in all varieties. This study showed that wheat varieties grown under identical growing conditions and management differ significantly in PC content and composition.

A New Type of Floral Oil from Malpighia coccigera (Malpighiaceae) and Chemical Considerations on the Evolution of Oil Flowers

New, partially acetylated dihydroxy fatty acids could be identified in the floral oil of Malpighia coccigera (Malpighiaceae): 7-OAc,3-OH 20:0, 7-OAc,3-OH 22:0, 9-OAc,3-OH 22:0, 9-OAc,5-OH 22:0, 3,9-diOAc 22:0, 9-OAc,3-OH 24:0, and 11-OAc,5-OH 24:0. The substitution patterns of all hitherto undescribed dihydroxylated and additionally identified monohydroxylated fatty acids are in agreement with a polyketide analogous biosynthesis. Intermediates may be 3-acetoxy fatty acids (C16, C18, and C20), known from flower secretions of other phylogenetically unrelated plant families. A possible relationship between plant epicuticular wax and floral oil biosynthesis is discussed. It may explain why an independent but convergent development of oil flowers and flower oils in unrelated plant families was possible.

Thermotropic Mesomorphism of a Model System for the Plant Epicuticular Wax Layer

As a model for the epicuticular wax layer of plant cuticular membranes, we have studied the phase behavior of 1-tetradecanol and 1-octadecanol and their binary mixtures between 5 and 70°C, using differential scanning calorimetry and Fourier transform infrared spectroscopy (FTIR). Both pure compounds show two exothermic phase transitions corresponding to a transformation from a liquid phase to a hexagonally packed solid phase (S HEX), which at lower temperatures transforms to an orthorhombically-packed solid phase (S ORT). On heating the S ORT solid a single endothermic transition with a transition enthalpy corresponding to the sum of the exothermic transition enthalpies is obtained. These transitions were also followed using FTIR spectroscopy in the CH 2-stretching (symmetric and asymmetric) and CH 2-rocking vibration modes. The FTIR spectra of the pure compounds in the liquid, S HEX, and S ORT phases were used to simulate experimental spectra in the phase transition regions. The simulations allowed us to estimate the molar fractions of each phase in the transition regions of the pure compounds. A phase diagram for the binary mixture of 1-tetradecanol and 1-octadecanol was obtained using differential scanning calorimetry and FTIR. FTIR studies on binary mixtures prepared from one perdeuterated component and the other nondeuterated permitted studying the thermotropic behavior of each component in the mixture independently. The mixture shows an eutectic behavior with an eutectic point between a molar fraction of octadecanol ( X 18) of 0.12 and 0.18 and a temperature of ∼32°C. Below 32°C, a binary mixture of solid phases, one an S ORT phase and the other an S HEX phase, coexist up to ∼25°C, below which both solid phases are S ORT phases. We discuss the possible relevance of this complex phase behavior in a simple binary mixture of two long-chain alkanols in the context of the far more complex phase behavior expected for the plant epicuticular wax layer.

Movement and regeneration of epicuticular waxes through plant cuticles.

Regeneration of plant epicuticular waxes was studied in 24 plant species by high-resolution scanning electron microscopy. According to their regeneration behaviour, four groups could be distinguished: (i) regeneration occurs at all stages of development; (ii) regeneration occurs only during leaf expansion; (iii) regeneration occurs only in fully developed leaves; (iv) plants were not able to regenerate wax. Wax was removed from the leaves with water-based glue and a liquid polymer, i.e. water-based polyurethane dispersion. In young leaves these coverings could not be removed without damaging the leaves. After a few days, waxes appeared on the surface of these polymer films, which still adhered to the leaves. It is concluded that waxes move through the cuticle in a process similar to steam distillation. This hypothesis could be further substantiated in refined in vitro experiments. Wax isolated from Eucalyptus globulus was applied to a filter paper, subsequently covered with a liquid polymer and fixed onto a diffusion chamber filled with water. The diffusion chamber was put into a desiccator. After 8-10 days at room temperature, crystals similar in dimensions and shape to in situ crystals appeared on the surface of the polyurethane film. This indicates that waxes in molecular dimensions move together with the water vapor that permeates through the polymer membrane. Based on these results, we propose a new and simple hypothesis for the mechanism of wax movement: the molecules that finally form the epicuticular wax crystals are moved in the cuticular water current.

Host finding and oviposition behavior in a chrysomelid specialist--the importance of host plant surface waxes.

Although several studies have elucidated the role of plant epicuticular waxes in host recognition and oviposition by herbivorous insects, there is little known about this subject in Chrysomelidae. In the present study, chemical cues for host recognition behavior and oviposition by the monophagous chrysomelid species Cassida stigmatica were investigated with special regard to surface waxes of the host plant. Tanacetum vulgare (Asteraceae). After hibernation in the soil, adults of this species must climb the plant's petioles, which emerge from the ground in spring. The response of adult C. stigmatica to contact and volatile cues from petioles and leaves of T. vulgare was investigated in a "stem arena," in which differently treated petioles and petiole dummies were offered. Volatile and contact cues of T. vulgare petioles served as well for host recognition. The contact cues were isolated from the petioles and leaves by hexane extraction and by cellulose acetate treatment, which removed cuticular waxes. The attractive volatiles were not extracted sufficiently by hexane. To examine the role of cuticular waxes of the host leaf surface in oviposition, female C. stigmatica were offered intact leaflets and leaflets from which cuticular waxes had been stripped by cellulose acetate treatment. Females did not discriminate between intact and stripped leaflets when only the upper leaf surfaces were offered. However. when the lower leaf surfaces that are generally used as oviposition sites were offered, C. stigmatica preferred to lay eggs on intact leaflets. We conclude that waxes of the lower leaf surface contain crucial information for oviposition in C. stigmatica.

A comparison of the composition of epicuticular wax from red raspberry ( Rubus idaeus L.) and hawthorn ( Crataegus monogyna Jacq.) flowers

Epicuticular waxes have been characterised from the flowers of raspberry and hawthorn, on both of which adult raspberry beetles ( Byturus tomentosus) can feed. The flower wax from both species had similar alkane profiles and also contained long-chain alcohols, aldehydes and fatty acids. The range of the carbon numbers detected for these classes of compounds was broadly similar in both but the relative amounts of each differed between species. Raspberry flower wax also contained fatty acid methyl esters, a group of compounds that has rarely been detected in plant epicuticular waxes, however, these were not observed in hawthorn flower wax. Long-chain alcohol-fatty acid esters with carbon numbers ranging from C36 to C48 were also detected in both plant species. However, an examination of their constituent acids indicated that in hawthorn the esters based on the C16 fatty acid predominated, whilst in raspberry flower wax, esters based on the C20 fatty acid were most abundant. Both species also contained pentacyclic triterpenoids, which accounted for, on average, over 16 and 48% of the total wax extracted from raspberry and hawthorn flowers respectively. In the former, ursolic and oleanolic acids accounted for over 90% of the pentacyclic triterpenes, whilst hawthorn flower wax, in addition to containing these acids, also contained high relative concentrations of both free and esterified α- and β-amyrins.

Oviposition by Plutella xylostella (Lepidoptera: Plutellidae) and effects of phylloplane waxiness.

Three approaches were used to investigate effects of host plant epicuticular waxes on oviposition site selection by Plutella xylostella (L.). In the first approach, oviposition on canola (Brassica napus L.) that had epicuticular wax reduced by application of a carbamate herbicide (S-ethyl dipropylthiocarbamate) was compared with oviposition on untreated control plants. A second approach compared oviposition on sibling strains of B. napus with different wax blooms (glossy and waxy), and a third approach compared oviposition by P. xylostella on parafilm that had been applied to glossy and waxy B. napus strains for transfer of leaf components. Significantly more eggs were deposited on herbicide-treated plants (with reduced epicuticular wax) than on untreated controls. Similarly, more eggs were deposited on glossy than on waxy sibling strains of B. napus. In parafilm assays significantly more eggs were deposited on treated than on untreated parafilm. Several mechanisms could explain the differences in attractiveness of surfaces with varying wax content as oviposition sites for P. xylostella, including visual, chemical, and tactile differences between substrates. These mechanisms are discussed.

Direct Access to Plant Epicuticular Wax Crystals by a New Mechanical Isolation Method.

A new method for the isolation of wax crystals from plant surfaces is presented. The wax-covered plant surface, e.g., a piece of a leaf or fruit, is brought into contact with a preparation liquid, e.g., glycerol or triethylene glycol, and cooled to ca. -100 degrees C. When the plant specimen is removed, the epicuticular wax remains embedded in the frozen liquid. After it warms up, the wax layer can be captured on appropriate carriers for further studies. This isolation method causes very little stress on the wax crystals; thus the shape and crystal structure are well preserved. In many cases it is possible, by choosing a preparation liquid with appropriate wettability, to isolate either the entire epicuticular wax layer or only discrete wax crystals without the underlying wax film. These crystals are well suited for electron diffraction studies by transmission electron microscopy and high resolution imaging by atomic force microscopy. The absence of intracuticular components and other impurities and the feasibility of the selective isolation of wax crystals enable improved chemical analysis and a more detailed study of their properties.

Classification and terminology of plant epicuticular waxes

Plant cuticles are covered by waxes with considerable ultrastructural and chemical diversity. Many of them are of great systematic significance. Waxes are an essential structural element of the surface and of fundamental functional and ecological importance for the interaction between plants and their environment. An extensive literature has been published since the introduction of scanning electron microscopy (SEM). Hitherto, the area has lacked a complete classification and terminology necessary as a standard for comparative descriptions. A refined classification and terminology of epicuticular waxes is therefore proposed based on high-resolution SEM analysis of at least 13 000 species, representing all major groups of seed plants. In total 23 wax types are classified. Thin wax films appear to be ubiquitous, while thicker layers or crusts are rare. The most prominent structures are local wax projections, which most probably result from self-assembly of wax molecules. These projections are supposed to be mainly of a crystalline nature and are termed crystalloids here. Among these, platelets and tubules are the most prominent types, while platelets arranged in parallel rows and stomatal wax chimneys are the most striking orientation and aggregation patterns. In addition, a comprehensive overview on the correlation between wax ultrastructure and chemical composition is given.

Classification and terminology of plant epicuticular waxes

Plant cuticles are covered by waxes with considerable ultrastructural and chemical diversity. Many of them are of great systematic significance. Waxes are an essential structural element of the surface and of fundamental functional and ecological importance for the interaction between plants and their environment. An extensive literature has been published since the introduction of scanning electron microscopy (SEM). Hitherto, the area has lacked a complete classification and terminology necessary as a standard for comparative descriptions. A refined classification and terminology of epicuticular waxes is therefore proposed based on high-resolution SEM analysis of at least 13 000 species, representing all major groups of seed plants. In total 23 wax types are classified. Thin wax films appear to be ubiquitous, while thicker layers or crusts are rare. The most prominent structures are local wax projections, which most probably result from self-assembly of wax molecules. These projections are supposed to be mainly of a crystalline nature and are termed crystalloids here. Among these, platelets and tubules are the most prominent types, while platelets arranged in parallel rows and stomatal wax chimneys are the most striking orientation and aggregation patterns. In addition, a comprehensive overview on the correlation between wax ultrastructure and chemical composition is given.

Organic geochemistry and coal petrology of Tertiary brown coal in the Zhoujing mine, Baise Basin, South China: 2. Biomarker assemblage and significance

The biomarker assemblage in the aliphatic fraction of the Tertiary brown coal from Zhoujing mine has been documented in detail. This aliphatic fraction consists of resin-derived diterpenoid and sesquiterpenoid hydrocarbons, long chain n-alkanes from vascular plant epicuticular waxes, and to a lesser extent terrigenous triterpenes. The huminite reflectance ( R 0) and biomarker parameters indicate the hydrocarbons in this brown coal are immature. Thus, resins and waxes of higher plants are particularly important for the generation of immature hydrocarbons from brown coal or lignite.

CRYSTALLINE STRUCTURE OF PLANT EPICUTICULAR WAXES DEMONSTRATED BY CRYOSTAGE SCANNING ELECTRON MICROSCOPY

SUMMARYThe epicuticular wax structure of several species was investigated, using plant material frozen rapidly in liquid nitrogen, and observed by means of a cryogenic specimen stage fitted to a scanning electron microscope. The wax was found to have a crystalline fine structure identical with that observed using conventional techniques for the preparation of plant surface specimens for transmission and scanning electron microscopy. The findings are discussed in relation to recent reports that the waxes are amorphous at low temperatures, and that the generally‐accepted crystalline fine structure of plant epicuticular waxes is an artefact of conventional specimen preparation procedures.

Organic matter of the troposphere—II. Natural background of biogenic lipid matter in aerosols over the rural western united states

Higher plant waxes are the predominant natural components in the lipid fractions (> C 15) of aerosols sampled over rural and oceanic regions. Hydrocarbon, fatty acid, ketone and fatty alcohol fractions of the lipids were characterized in terms of their contents of homologous compound series and specific biogenic molecular markers. Paniculate samples from the rural western United States have been analyzed and compared with samples from urban Los Angeles and remote areas over the Atlantic Ocean. The samples from rural sites contained predominantly vascular plant wax and lesser amounts of higher plant sterols and resin residues. Urban samples and, to varying degrees, some rural samples contained primarily higher weight residues of petroleum products. The loadings of hydrocarbons derived from higher plant waxes ranged approximately from 10 to 160 ng m −3 of air (for fatty acids, 10–100 ng m −3 and for fatty alcohols, 10–200 ng m −3). Higher molecular weight lipids (i.e. plant epicuticular wax, terpenes, etc.) from flora comprise a significant component of the organic carbon in rural aerosols. Primary biogenic residues are major components of aerosols in all areas and they are important components in the global cycling of organic carbon.

Structural determination of secondary alcohols from plant epicuticular waxes

Structural determination of secondary alcohols from plant epicuticular waxes

ULTRASTRUCTURE AND RECRYSTALLIZATION OF PLANT EPICUTICULAR WAXES

SUMMARYA wick feed method was used to recrystallize a range of different plant epicuticular waxes from solutions in organic solvents. Using the scanning electron microscope the ultrastructure of the recrystallized wax from each species was compared with that of the wax on the corresponding intact plant surface and in many cases was found to be similar. It is concluded that the crystal structure of epicuticular waxes is greatly influenced by their inherent chemical and physical properties rather than by properties of the underlying cuticular membrane or by any mechanism of extrusion or transport of the wax to the plant surface.

Method for recrystallizing selected components of plant epicuticular waxes as surfaces for the growth of micro-organisms

Method for recrystallizing selected components of plant epicuticular waxes as surfaces for the growth of micro-organisms