FTIR spectroscopy of synthesized racemic nonacosan-10-ol: a model compound for plant epicuticular waxes

As there are no published graphically presented, detailed IR spectra of nonacosan-10-ol (occurring naturally and widely in plant epicuticular waxes of nanotube form), near IR FTIR spectroscopy (fundamentals, overtones and combinations) has been performed on laboratory synthesized racemic nonacosan-10-ol, as a crystalline solid on Mylar and polypropylene substrates. Room temperature, in vacuo data are presented graphically, in full, and show evidence of extensive hydrogen bonding, an orthorhombic perpendicular subcell, a methylene wagging progression, diagnostic of all-trans conformational order, and Fermi resonance. Moderate or stronger anharmonicity is confirmed. Detailed discussion, quantitative in parts, is given of the observed spectral features, especially as to how they inform crystal structure and molecular conformation, and assignments given for some of the features. The results will serve as a reference for future IR studies of the natural epicuticular wax nanotube form of (S)-nonacosan-10-ol.     

Chemodiversity of nonacosan-10-ol and n-alkanes in the needle wax of Pinus heldreichii

This is the first report of individual variability and population diversity of the contents of nonacosan-10-ol and n-alkanes in the needle cuticular waxes of Bosnian pines originated from Montenegro, regarded as Pinus heldreichii var. leucodermis, and from Serbia, regarded as P. heldreichii var. pan?i?i. The amount of nonacosan-10-ol varied individually from 27.4 to 73.2% (55.5% in average), but differences between the four investigated populations were not statistically confirmed. The size of the n-alkanes ranged from C(18) to C(33). The most abundant n-alkanes were C(23), C(27), and C(25) (12.2, 11.2, and 10.8% in average, resp.). The carbon preference index (CPI) of the n-alkanes ranged from 0.8 to 3.1 (1.6 in average), while the average chain length (ACL) ranged from 20.9 to 26.5 (24.4 in average). Long-chain and mid-chain n-alkanes prevailed (49.6 and 37.9% in average, resp.). It was also found that the populations of P. heldreichii var. leucodermis had predominantly a narrower range of n-alkanes (C(18)-C(31)) than the trees of the variety pan?i?i (C(18)-C(33)). Differences between the varieties were also significant for most of the other characteristics of the n-alkane pattern (e.g., most abundant n-alkanes, CPI, ACL, and relative proportion of short-, mid-, and long-chain n-alkanes). The principle component and cluster analyses of eleven n-alkanes confirmed the significant diversity of these two varieties.     

Epicuticular crystals of nonacosan-10-ol: In-vitro reconstitution and factors influencing crystal habits

The primary aerial surfaces of plant species from many families (e.g. Pinaceae, Liliaceae, Ranunculaceae, Papaveraceae) are covered by epicuticular tubules 5–20 μm long and 0.5 μn in diameter. The composition, mechanism of growth and molecular structure of this type of epicuticular aggregates have been studied. Pure nonacosan-10-ol extracted from Picea pungens needle surfaces formed, in vitro, tubular crystals like those occurring in vivo. This crystal habit was obtained irrespective of the type of solvent or substratum, if the solvent was evaporated within minutes. This shows that tubules of nonacosan-10-ol are formed in the kinetic regime of crystallization (limited by the diffusion of molecules from the solution to the crystal surface). Slow evaporation of the solvent or crystallization from the melt resulted in rhombic scales. These planar crystals represent the thermodynamic, stable modification of native nonacosan-10-ol. Homologous impurities in natural nonacosan-10-ol (3–14%) had no effect on the formation of the tubules. However, racemic nonacosan-10-ol invariably crystallized in scales. The phase behaviour of mixtures of natural nonacosan-10-ol and its synthetic racemate as well as synthetic (S)-nonacosan-10-ol provided evidence for the presence of the pure (S)-enantiomer on plant surfaces. The findings are discussed in terms of the mechanisms leading to epicuticular tubules consisting of nonacosan-10-ol and their molecular structure. Crystal structures for the pure enantiomer and the racemate of nonacosan-10-ol are proposed. It is concluded that the principles responsible for the formation of tubules are both the special molecular geometry of the naturally occurring (S)-nonacosan-10-ol and the mobility barrier of the plant cuticle. Further specific biological processes are not necessary for the formation of (S)-nonacosan-10-ol tubules. The alterations of epicuticular structures during ageing or the impact of pollutants are explained as spontaneous transitions between two crystal modifications of (S)-nonacosan-10-ol.     

Inhibition of epicuticular wax deposition on cabbage by ethofumesate

The weight of epicuticular wax on the surface of cabbage (Brassica oleracea var. Capitata `Market Prize') leaves was reduced by soil treatments of ethofumesate (2-ethoxy-2,3-dihydro-3,3-dimethyl-5-benzofuranyl methanesulfonate) and EPTC (S-ethyl dipropylthiocarbamate). Separation of epicuticular wax into major components by gas-liquid chromatography indicated that ethofumesate decreased the deposition of n-nonocosane and n-nonocosan-15-one on cabbage leaves but increased the deposition of a minor component, the long chain waxy esters. EPTC was less inhibitory to n-nonocosan-15-one deposition than was ethofumesate. EPTC did not increase long chain waxy ester deposition. Scanning electron micrographs revealed that ethofumesate almost totally eliminated the epicuticular wax on cabbage leaves while EPTC only diminished it. Cuticular transpiration was increased by ethofumesate but not by EPTC. Ethofumesate appears to be a more potent inhibitor of epicuticular wax deposition than EPTC.     

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

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

Biosynthesis of the beta-diketones of barley spike epicuticular wax.

[1-¹⁴C]acetate and [2-¹⁴C]acetate were incorporated into the β-diketones of barley spike epicuticular wax via the peduncle. Utilizing column chromatography with dry copper acetate, the β-diketones were isolated and the labeling pattern in the hentriacontan-14, 16-dione determined after its degradation. A modified iodoform procedure was used to give myristic and palmitic acids. Radio-gas chromatography was then performed on the products of chemical α-oxidation of the separated fatty acids. This procedure, in effect, gave the specific activity of every carbon atom of hentriacontan-14,16-dione except carbon-1 to carbon-5 (from myristic acid) and carbon-27 to carbon-31 (from palmitic acid) for each labeled substrate. The specific activity of carbon-15 was determined by an indirect method. On the basis of these data it is suggested that the hentriacontan-14,16-dione is synthesized from the carbon-31 end of the molecule by elongation as follows. C₂ units are added, perhaps to a mixture of short chain precursors, to give a chain with 12 carbon atoms. This chain is then elongated to one with 16 carbon atoms so that the four added carbon atoms are uniformly labeled. Following this, the chain with 16 carbon atoms is elongated with C₂ units to give the complete molecule. Possibly some change in mechanism occurs in this last elongation process when the chain is 22 carbon atoms long. Barley spike wax β-diketones contain about 2% nonacosan-13, 15-dione which seems to be synthesized in an analogous manner.     

Studies on stomatal function, epicuticular wax and stem-root transition region of polyethylene glycol-treated and nontreatedin vitro grape plantlets

Summary Scanning electron microscopy, light microscopy, and gravimetric analysis was used to evaluate stomatal function, epicuticular wax, and the stem-root transition region of grape (Vitis sp. ‘Valiant’) plantlets grownin vitro, polyethylene glycoltreatedin vitro, and greenhouse-grown plants. Scanning electron microscopic studies of leaf surfaces ofin vitro-grown plants showed widely open stomata as compared to leaf stomata of polyethylene glycol-treatedin vitro-cultured and greenhouse-grown plants. Ultrastructurally, leaf epicuticular wax ofin vitro plants was less dense than in their polyethylene-treated and greenhouse counterparts. Quantitatively,in vitro-grown plants had reduced epicuticular was as compared to polyethylene glycol-treated and greenhouse-grown plants. Light microscopic studies showed no obvious differences in the vascular connections in the stem-root transition region ofin vitro-cultured, polyethylene glycol-treatedin vitro-cultured, and greenhouse-grown plants. It is therefore likely that the rapid wilting and desiccation observed after transplantingin vitro grape plantlets is due to their defective stomatal function and reduced epicuticular wax and may not be due to poor water transport associated with vascular connection.     

The skimmiwallinols--minor components of the epicuticular wax of Cocos nucifera

Four new skimmiwallinol derivatives, isoskimmiwallinol acetate (4), skimmiwallinol acetate (5), isoskimmiwallinone (6) and skimmiwallinone (7), along with the previously isolated major components isoskimmiwallin (2) and skimmiwallin (3), have been found in the wax extract of pinnae from Cocos nucifera. Metabolites 4 and 5 were obtained as an inseparable mixture and identified by analysis of spectroscopic data and chemical correlation with 2 and 3, respectively. Metabolites 6 and 7 were identified by GC co-injection with the corresponding synthetic derivatives prepared from 4 and 5.     

Effect of epicuticular wax isolation method on alkane distribution pattern

The alkane distribution pattern in epicuticular wax isolated by a single immersion of leafy material in light petroleum does not differ significantly from wax obtained by two consecutive immersions at room temperature. However, was isolated by immersion in boiling light petroleum gives in some cases an alkane pattern different from the wax isolated by immersion at room temperature.     

Chemical composition of the epicuticular wax from the fruits of Eucalyptus globulus

The chemical composition of the epicuticular wax from the fruits of Eucalyptus globulus was studied by GC-MS before and after alkaline hydrolysis. The wax had two main components, ursolic acid and tritriacontan-16,18-dione, together with several other triterpenic acids. After alkaline hydrolysis, a large increase in the amounts of triterpenic acids and fatty acids (particularly in hexadecanoic acid) was observed, suggesting that these components were present predominantly in esterified forms in the fruit wax. Six compounds were isolated from the fruits by preparative chromatography, and were identified as 8-desmethyleucalyptin, sesamin, tritriacontan-16,18-dione, ursolic acid, 3beta-hydroxyurs-11-en-13beta(28)-olide (ursolic acid lactone) and 3beta,11alpha-dihydroxyurs-12-en-28-oic acid, the latter of which was identified for the first time.     

Population variability of nonacosan-10-ol and n-alkanes in needle cuticular waxes of Macedonian pine (Pinus peuce GRISEB.)

This is the first report on population variability of nonacosan-10-ol and n-alkanes in needle epicuticular waxes of Macedonian pine (Pinus peuce GRISEB.) Hexane extracts of needle samples, originating from two natural populations in Montenegro (Zeletin and Sjekirica) and from one population in Serbia (Mokra Gora) were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The amount of nonacosan-10-ol varied individually from 41.3 to 72.31% (average 55.9%), with the Sjekirica population being statistically divergent (64.4% on average). The results showed n-alkanes in epicuticular waxes ranging from C?? to C??. The most abundant alkanes were C??, C??, C??, and C?? (15.5, 11.1, 10.6, and 10.5% on average, resp.). The carbon preference index of Pinus peuce ranged from 1.0 to 4.3 (1.9 on average). Average chain length ranged from 18.4 to 27.7 (average 25.7). A high level of inidividual quantitative variation in all of these hydrocarbon parameters was also detected. These results were compared with published data on other species from the Pinus genus.     

Effects of leaf epicuticular wax on the movement, foraging behavior, and attack efficacy of Diaeretiella rapae

Plant morphological characters can affect the foraging abilities of natural enemies. Heavy wax blooms have been shown to impede predators searching for herbivores on various species in the genus Brassica (Brassicaceae). This study determined whether epicuticular wax affected the foraging behavior of the braconid wasp Diaeratiella rapae (M’Intosh) (Hymenoptera: Braconidae) as it searched for its aphid host Brevicoryne brassicae L. (Homoptera: Aphididae) on the leaves of cauliflower varieties with heavy or light wax blooms. Wasps on the variety with a heavier wax bloom foraged more slowly, groomed more often and for longer periods of time, fell from the leaves more often, took longer to find colonies of aphids, and attacked them at a lower rate than wasps foraging on the variety with a lighter wax bloom. When epicuticular wax was removed from the leaves, the wasp's foraging efficiency and efficacy improved significantly on the cauliflower variety with a heavy wax bloom. The amount of epicuticular wax present on a leaf was shown to impede the ability of a parasitoid to forage, locate, and attack its host.     

Effects of light and temperature on the composition of epicuticular wax of barley leaves

The amount of wax/cm² on expanding primary leaves of Bonus barley depends on both the photo- and thermoperiods in which the seedlings are grown. With a temperature cycle of 15–10°, transfer of dark grown leaves to the light stopped leaf expansion and after 24 hr yielded 2·5 times more wax/cm² than is characteristic for light grown leaves. This demonstrates that wax synthesis and extrusion is not directly correlated with leaf expansion. The relative amounts of the wax classes formed by the decarboxylation pathways (< 1%), the reductive pathways (89%) or only by elongation (10%) are the same in light and dark. Within the reductive pathways, however, light stimulates aldehyde formation. Both environmental parameters can strongly influence the chain length composition of the wax classes. In the light one chain length or one group of chain lengths dominates a given wax class. In the dark two prominent chain lengths or groups thereof are found. The major chain length in these two groups differs by two or more carbons.     

Effect of leaf age on epicuticular wax alkanes in Rhododendron

Alkane distribution pattern in epicuticular wax of Rhododendron fortunei cv Admiral Piet Hein leaves remains unchanged from the age of 6 weeks to abscission.     

Complex biopolymeric systems at stalk/epicuticular wax plant interfaces: A near infrared spectroscopy study of the sugarcane example

Naturally occurring macromolecules present at the epicuticular wax/stalk tissue interface of sugarcane were investigated using near infrared spectroscopy (NIRS). Investigations of water, cellulose, and wax‐cellulose interrelationships were possible using NIRS methods, where in the past many different techniques have been required. The sugarcane complex interface was used as an example of typical phenomena found at plant leaf/stalk interfaces. This detailed study showed that sugarcane cultivars exhibit spectral differences in the CH_n, water OH, and cellulose OH regions, reflecting the presence of epicuticular wax, epidermis, and ground tissue. Spectrally complex water bands (5276 cm^?1 and 7500–6000 cm^?1) were investigated via freeze‐drying experiments which revealed sequentially a complex band substructure (7500–6000 cm^?1), a developing weak H‐bonding system (～7301 cm^?1), and strong H‐bonding (～7062 cm^?1) assigned to water—cellulose interactions. Principal component analysis techniques clarified complex band trends that developed during the desorption experiment. Bands from wax‐free stalk were minimized in the 4327–4080 cm^?1 region (C? H_n vibrational modes associated with long chain fatty compounds), while bands from the stalk tissue (particularly lignin and moisture) became more pronounced. This work is a comprehensive guide to similar studies by scientists involved in a variety of plant and fiber research fields. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 642–651, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

p-Hydroxybenzaldehyde as a major constituent of the epicuticular wax of seedling Sorghum bicolor

p-Hydroxybenzaldehyde is present in concentrations up to 30% in the wax of Sorghum bicolor seedlings. It is highly deterrent to locusts, reducing their normal feeding by 90%.