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AOBPreview originally published online on July 3, 2007
Annals of Botany 2007 100(2):225-231; doi:10.1093/aob/mcm120
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© The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Epicuticular Wax Crystals of Wollemia nobilis: Morphology and Chemical Composition

Simona Dragota and Markus Riederer*

Universität Würzburg, Julius-von-Sachs-Institut für Biowissenschaften, D-97082 Würzburg, Germany

* For correspondence. E-mail riederer{at}uni-wuerzburg.de

Received: 22 December 2006    Returned for revision: 16 April 2007    Accepted: 26 April 2007    Published electronically: 3 July 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

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.

Key words: Wollemia nobilis, epicuticular wax, nonacosan-10-ol, nonacosane diols, SEM


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
The cuticle of terrestrial plants is a multifunctional interface between plant and environment, containing a polyester-like biopolymer cutin impregnated and covered by waxes (Riederer and Müller, 2006). The waxes are both embedded in the polymer matrix (intracuticular waxes) and deposited on the surface (epicuticular waxes) of primary plant organs. The physico-chemical characteristics of the cuticular waxes (Schönherr, 1976, 1982; Schönherr and Riederer, 1989; Bianchi, 1995; Riederer and Schreiber, 1995) contribute to important physiological and ecological properties of the plant cuticle (e.g. control of transpiration, hydrophobicity, optical properties, and interactions with chemicals and with other organisms). In most cases, plant waxes are a complex mixture of long-chain aliphatic components, such as n-alkanes, fatty acids, alcohols, aldehydes, ketones or n-alkyl esters (Müller and Riederer, 2005, and literature cited therein). Additionally, various amounts of cyclic compounds such as pentacyclic triterpenoids or flavonoids have been reported to be components of cuticular waxes (Barthlott and Wollenweber, 1981; Baker, 1982; Holloway, 1984; Walton, 1990). Methods for selective mechanical removal of epicuticular wax films and hence the direct analysis of surface composition have recently become available (Ensikat et al., 2000; Jetter et al., 2000; Jetter and Schaeffer, 2001). Due to their chemistry and microstructure, epicuticular wax crystals constitute a superficial micro-structured layer that is ultra-hydrophobic and which thereby increases water repellence and reduces adhesion of contaminating particles (Barthlott and Neinhuis, 1997).

Wollemia nobilis is a remarkable evergreen tree of the conifer family Araucariaceae, discovered in 1994 by David Noble in the Wollemi National Park, located in the Blue Mountains, 150 km north-west of Sydney, Australia (Jones et al., 1995). At present, this species is known only from a stand with approximately 40 adult trees (Jones et al., 1995) and several hundred seedlings (Offord, 1996), and the fossil pollen of Dilwynites granulatus (Macphail et al., 1995). Wollemia nobilis is one of the world's rarest plants, dating back to the ‘time of dinosaurs’. Morphological features (Hill, 1997; Burrows, 1999; Burrows and Bullock, 1999; Offord et al., 1999; Burrows et al., 2003), affiliation to cretaceous fossils (Macphail et al., 1995; Chambers et al., 1998), DNA analyses (da Silva, 1997; Gilmore and Hill, 1997; Setoguchi et al., 1998; Stefanovic et al., 1998) and studies on the leaf essential oil (Brophy et al., 2000) have demonstrated that Wollemia is a new monotypic genus in the family Araucariaceae, next to Agathis and Araucaria. Studies on chromosome number, karyotype and DNA C-values have revealed similarity to other species in the Araucariaceae (Hanson, 2001). The extremely low genetic variation of this tree is an interesting characteristic that has recently been reported (Peakall et al, 2003). Studies on leaf anatomy of W. nobilis have revealed differences between juvenile and adult leaves (Burrows and Bullock, 1999).

The objective of the present study was to investigate the morphology of the epicuticular leaf waxes of Wollemia nobilis using scanning electron microscopy and to study their chemical composition using gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy. As there are two leaf types in this plant species, analyses were done for both types and for the adaxial and abaxial leaf surfaces, respectively.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Plant material
Leaves sampled from a Wollemia nobilis W. Jones, K. Hill & J. Allen (Araucariaceae) tree grown in the Botanical Garden of the University of Wuerzburg were used in this study. The 4–5 year-old seedling was kindly donated to the Botanical Garden by Fa. Kientzler, Gensingen, Germany.

Scanning electron microscopy (SEM) and image analyses
Wollemia nobilis leaves were examined at low temperature in a Hitachi S-4700 cold field emission scanning electron microscope (FESEM), which was interfaced to a Gatan Alto 2500 cryotransfer system. Leaf pieces of 1 cm were attached to a stub (aluminium stub, length 3 mm, diameter 10 mm) and secured in a Gatan Alto 2500 dovetail specimen holder using a graphite/PEG/PPG adhesive (agar colloidal graphite/polyethylene glycol/polypropylene glycol). The adhesive was warmed on a hotplate until it melted at 55 °C. A drop of the molten adhesive was placed on the specimen stub and allowed to cool to 35 °C before attaching the leaf. The temperature was monitored using an RS Components Type K thermocouple. The adhesive was allowed to set fully before locating the specimen holder onto a vacuum transfer device (VTD). The VTD was transferred to the slushing station of the Gatan Alto 2500 cryotransfer system and the sample was plunge-cooled in slushy liquid nitrogen. It was subsequently transferred, under vacuum, to the sample stage of an Alto 2500 preparation chamber. The stage temperature was raised to –135 °C and the chamber pressure increased to ~ 4 x 10–4 mbar using argon gas, prior to sputter-coating with platinum for 100 s at 300 V/10 mA. The coated specimen was transferred into the Hitachi S-4700 FESEM chamber and located on the cold stage (also maintained at – 135 °C ) for observation at an accelerating voltage of 2·0 kV and a working distance of 12 mm using mixed upper and lower secondary electron detectors. Images were recorded digitally using the Quartz PCI imaging system.

Preparation of wax samples for GC studies
Preliminary experiments were performed to establish a method for a selective probing of the epicuticular waxes. The chemical properties of a solvent (polarity and density), its volume and temperature together with the contact time between solvent and plant surface were found to represent crucial parameters that have to be optimized for a selective extraction. CHCl3 (purity 99 %; Roth, Karlsruhe, Germany) was shown to be appropriate and to give reproducible results. Unfortunately, by dipping the plant leaves in CHCl3 for 30 s at room temperature, contamination of the epicuticular wax extract with small amounts of internal lipids and larger amounts of leaf oil and resin components was observed. Therefore, the contact time was reduced to minimize possible contamination. The solvent was applied for 1 s to the adaxial and abaxial sides of the leaves using a soft paintbrush moistened with CHCl3. Prior experiments had established how often the leaf surface can be brushed before contamination of the epicuticular wax fraction occurred. A clean paintbrush was first dipped in CHCl3 for 30 s and the selected section of the leaf surface was brushed once within 1 s. The extracted wax was than removed from the paintbrush hairs by re-dipping in the solvent used for extraction for 30 s. In addition, a blank sample was prepared by dipping the paintbrush in fresh solvent for 60 s to establish the possible impurities caused by the paintbrush hairs. The solutions resulting from brushing two juvenile and two adult leaves, respectively (one leaf side was brushed for each extraction), were dried over Na2SO4, filtered off, and the solvent was completely removed under a gentle stream of nitrogen at 50 °C. In order to enhance GC separation of the substance classes containing hydroxyl groups, the corresponding trimethylsiloxy derivatives were prepared by treatment of the remaining dry residue with N,O-bis(trimethylsilyl)-trifluoroacetamide in excess (BSTFA; Macherey-Nagel, Düren, Germany) at room temperature. After approx. 2 h CHCl3 was added to the reaction mixture to dilute the sample to a concentration suitable for GC analysis.

In addition to this method for epicuticular wax removal, a polymer film of gum arabic was used. Commercial gum arabic (Roth, Karlsruhe, Germany) was extracted exhaustively with hot chloroform to remove any soluble lipids, and residual organic solvent was allowed to evaporate completely. An aqueous solution of pre-extracted gum arabic (50 %) was applied to the adaxial or abaxial leaf surface using a small paintbrush. After approx. 30 min, the dry and mechanically robust polymer film was peeled off and transferred into a vial containing a two-phase system consisting of chloroform and water (1:2, v/v). Five and seven mechanical wax removals were performed from the adaxial and abaxial side, respectively, and the dried polymer films were collected into the same vial for each leaf side. Preliminary experiments had been performed to establish the number of removals necessary for isolating the epicuticular wax quantitatively from each leaf side. The chloroform phase, which contained the cuticular wax, was isolated using a separatory funnel, dried over anhydrous Na2SO4, filtered off and treated with BSTFA prior to GC analysis. Although special care was taken to avoid contamination through surface lesions, several terpenoids (kaur-16-ene, abietic acids or pimaric acids) and medium-chain fatty acids (saturated C16, C18; unsaturated C18) were found in the samples. Kaur-16-ene is an important component of W. nobilis leaf oil (Brophy et al., 2000) whereas abietic and pimaric acids are resin components of W. nobilis. Comparison with the data obtained after brushing the leaf surface revealed that the compounds mentioned above are very likely not components of the epicuticular waxes of W. nobilis leaves and consequently are not treated further in the present investigation.

Gas chromatographic analyses
GC analyses were carried out using a gas chromatograph (5890 series II, Hewlett Packard, Avondale, PA, USA) equipped with a flame ionization detector (FID) and a pressure-programmable on-column injector. Wax constituents were separated on a fused silica column DB-1 (length 30 m, diameter 320 µm, film thickness 0·1 µm). Chromatographic conditions were as follows. Temperature programme: injection at 50 °C and held for 2 min at 50 °C, increased by 10 °C/min to 220 °C, increased by 1 °C/min to 240 °C, increased by 10 °C/min to 320 °C and held for 15 min at 320 °C; pressure programme (inlet pressure of the hydrogen carrier gas): injection at 50 kPa and held for 5 min at 50 kPa, increased by 3 kPa/min to 150 kPa and held for 40 min at 150 kPa. Wax components were identified by GC–MS (GC 6890 N equipped with a 5973 mass selective detector; Agilent Technologies) using the same chromatographic conditions as described above, with the exception of using helium instead of hydrogen as carrier gas. For a quantitative wax analysis, GC–FID experiments were performed using n-tetracosane as internal standard and the surface area analysed was measured digitally by scanning photocopies of the leaves.

Preparation of wax samples for NMR studies
Waxes used for NMR studies were extracted by immersing 15 juvenile leaves of W. nobilis for 30 s in CDCl3 (10 mL, Euriso-top, Gif-sur-Yvette, France). After slow partial evaporation of the solvent (up to approx. 1 mL) at room temperature 1H, 13C NMR experiments with the resulting solution were performed on a Bruker Avance 500 spectrometer (500·13 MHz, 22 °C). Chemical shifts (ppm) were determined relative to internal CHCl3 (1H, {delta} 7·24; CDCl3), CDCl3 (13C, {delta} 77·0; CDCl3). The analysis and assignment of the 1H NMR data was supported by 1H,13C correlation experiments and the assignment of the 13C NMR data was supported by DEPT 135 experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 
Wollemia nobilis has two types of leaves: juvenile leaves with a bright, apple-green colour arranged in two opposite rows, and adult leaves with a deeper bluish-green colour arranged helically around the twig (Fig. 1).


Figure 1
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  		FIG. 1. Juvenile leaves (left) and adult leaves (right) of W. nobilis.

 
Low-temperature scanning electron microscopy was used for the characterization of W. nobilis epicuticular wax morphology. The crystalline microstructure of the wax and its distribution on the leaf surface were studied on the adaxial and abaxial sides of juvenile and adult leaves (Figs 2–4). Wollemia nobilis leaves are covered with a layer of crystalline tubular waxes that is fairly dense, especially on the abaxial side of the leaves. The scanning electron micrographs reveal that on the abaxial side tubular crystals are more abundant than on the adaxial side. Stomata with epistomatal cavities filled with tubular wax are found only on the abaxial area of the leaf (Fig. 2). Macroscopically, the abaxial side of the leaves has a whitish appearance due to light reflected from the dense coverage by tubular wax, while the adaxial side is glossy.


Figure 2
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  		FIG. 2. Low-temperature FESEM images of waxy surfaces of W. nobilis leaves. (A, C) Adaxial side of a juvenile leaf; (B, D) abaxial side of a juvenile leaf; (E, G) adaxial side of an adult leaf; (F, H) abaxial side of an adult leaf. Scale bars = 200 and 20 µm as indicated.

 
Some differences in wax deposition could be observed between juvenile and adult leaves (Fig. 3). Tubular crystals were slightly deteriorated especially on the adaxial surface of adult leaves. This change of micro-morphology is related to the distribution of the tubules and the presence of platelet-like aggregates lying flat on the substrate. Another type of platelet occurs on the abaxial side of adult leaves.


Figure 3
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  		FIG. 3. Low-temperature FESEM images of waxy surfaces of W. nobilis leaves. (A) Adaxial side of a juvenile leaf; (B) abaxial side of a juvenile leaf; (C) adaxial side of an adult leaf; (D) abaxial side of an adult leaf. Scale bars = 3 µm.

 
In all leaf types and on both the adaxial and abaxial surfaces, the epicuticular tubules are randomly orientated relative to the substrate, with the orientation spanning from parallel to vertical in relation to the leaf surface. In some cases, the tubules exhibit a pronounced helicity (Fig. 4A). Some of the tubular openings show loose ends of helically wound ribbons, suggesting that the wax tubules are formed by a clockwise circular growth of rodlets. Curled and straight rodlets could be observed in particular on the abaxial side of the leaves, perhaps representing different states of wax formation or different deterioration states of the tubules. At their endings some tubules show curved ribbons, which curl up into new tubules that grow in a different direction (Fig. 4B).


Figure 4
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  		FIG. 4. Low-temperature FESEM images of the abaxial side of a juvenile leaf of W. nobilis: (A) scale bar = 1 µm; (B) scale bar = 2 µm.

 
Quantitative determination of epicuticular wax mechanically removed with gum arabic yielded approx. 20 µg cm–2 from the adaxial and approx. 33 µg cm–2 from the abaxial surface of a juvenile leaf. Approximately 27 µg cm–2 of epicuticular wax was removed from the upper and approx. 35 µg cm–2 from the lower surface of an adult leaf. These results suggest that the epicuticular wax coverage of the abaxial side in both leaf types is somewhat higher than that of the adaxial side.

Gas chromatographic analyses of the epicuticular waxes revealed that the wax of W. nobilis leaves contains several aliphatic compound classes and some particularly abundant major compounds. The GC–MS studies performed with wax solutions (after chemical extraction and mechanical removal) established that the asymmetric secondary alcohol nonacosan-10-ol is the main component of the epicuticular waxes of W. nobilis. The corresponding trimethylsilyl derivative showed typical molecular mass fragments of 481, 369 and 229 m/z. Nonacosan-10-ol is not the single component of its class, as very small amounts of pentacosan-10-ol and heptacosan-10-ol were also detected. According to GC–MS analyses, nonacosan-10-ol together with its bifunctional analogues (nonacosane-4,10-, –5,10-, –7,10-, –10,13- and –10,16-diols) and odd-numbered n-alkanes (mainly C31, C33 and C35) were the principal wax constituents. The remaining epicuticular wax is made up of long-chain aliphatic constituents (such as primary alcohols, fatty acids or alkyl esters) but further discussion in this paper will be focussed only on the two major compound classes.

After epicuticular wax extraction using the paintbrush technique, GC analyses demonstrated that there are differences between the adaxial and the abaxial wax composition. The epicuticular wax on the adaxial side of the leaves is made up of nonacosan-10-ol (approx. 48 %), nonacosane diols (approx. 12 %) and n-alkanes (C31, C33, and C35; approx. 22 %), whereas the epicuticular wax composition of the lower side of the leaves contains nonacosane-10-ol (approx. 53 %), nonacosane diols (approx. 18 %) and n-alkanes (C31, C33, and C35; approx. 12 %). The juvenile and adult leaves have a similar distribution of these three compound classes between the two leaf sides. Comparisons with the relative composition obtained after mechanical removal with gum arabic revealed similar results with small differences as to the percentage of the compound classes mentioned.

The 1H and 13C NMR data (solvent CDCl3) unequivocally demonstrated the presence of nonacosan-10-ol as the main component of the extracted waxes. 1H,13C HMQC NMR experiments clearly indicate the correspondence of the 1H NMR resonance signals with the 13C NMR resonance signals of nonacosan-10-ol (see Fig. 5). 1H NMR (CDCl3, 22 °C, 500·13 MHz): 0·86 (t, 3JHH = 6·9 Hz, 6 H, CH3), 1·18–1·25 (m, 4 H, CH3CH2CH2), 1·21–1·27 (m, 4 H, CH3CH2), 1·23 (br s, 36 H, CH2), 1·23–1·32 and 1·36–1·42 (m, 4 H, CHCH2CH2), 1·37–1·43 (m, 4 H, CHCH2), 3·51–3·57 (m, 1 H, CH), OH (not detected). 13C NMR (CDCl3, 22 °C, 125·8 MHz): {delta} 14·1 (CH3), 22·7 (CH3CH2), 25·7 (CHCH2CH2), 29·4 (CH3CH2CH2CH2), 29·7 (CH2), 31·9 (CH3CH2CH2), 37·5 (CHCH2), 72·1 (CH).


Figure 5
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  		FIG. 5. Partial spectra obtained in a 1H,13C HMQC NMR experiment with wax solution of juvenile leaves of W. nobilis after extraction within 30 s with chloroform (500·13 MHz, CDCl3, 23 °C). Top: 1H NMR resonance signals. Left: 13C NMR resonance signals.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 LITERATURE CITED
 
The leaves of Wollemia nobilis are covered with tubular epicuticular crystals (Figs 2–4). The well-known correlation between morphology and chemical composition of epicuticular waxes is confirmed in the case of W. nobilis, where high relative amounts of the secondary alcohol nonacosan-10-ol (whose identity was confirmed by solution NMR studies, 1H, 13C) together with nonacosane diols coincide with the presence of tubular crystalline aggregates. Several prior studies have demonstrated that this type of tubular epicuticular wax crystal is made up of nonacosan-10-ol (Fuhrhop et al., 1994; Riederer and Jetter, 1994a, 1994b; Riederer et al., 1996; Barthlott et al., 1998, 2000; Herédia et al., 2003; Jetter et al., 2006; Koch et al., 2006). Tube-type waxes containing substantial amounts of nonacosane-10-ol are dominant in the Gymnosperm genera (e.g Ginkgo, Taxus, Picea, Pinus or Abies) and have also been found in the Angiosperm genera (e.g. Chelidonium majus, Papaver somniferum, Prunus domestica or Rosa spp.) (Jeffree, 2006).

The low-temperature scanning electron micrographs of the adaxial and abaxial sides of the W. nobilis leaves clearly demonstrate that the abaxial surface is more densely covered by wax tubules than the adaxial surface. This feature correlates well with the results of the qualitative and quantitative wax analyses. The amount of epicuticular wax removed from the abaxial leaf side is higher than that obtained from the adaxial one. In addition, the distribution of the main component classes shows that the relative amounts of the tubule-forming compounds nonacosane-10-ol and nonacosane diols are higher on the abaxial leaf side in comparison with that on the adaxial leaf side.

The helicity of the tubules, which has been attributed to the chirality of the naturally occurring nonacosan-10-ol (Fuhrhop et al., 1994; Riederer and Jetter, 1994a), was demonstrated very clearly in this study. The tubules exhibit helical striation with an approx. 100 nm period (Fig. 4). The striation was less pronounced with the tubules found on the adaxial sides, which may be due to the interference of n-alkanes that are more abundant on these leaf surfaces.

In general, there were no significant differences in the epicuticular wax composition between the juvenile and the adult leaves. The contribution of nonacosane-10-ol, nonacosane diols and n-alkanes to the total epicuticular wax content is very similar in both leaf types.

In conclusion, the results presented here provide a first insight into the fine structure and chemical nature of the epicuticular wax crystals found on the surfaces of W. nobilis leaves. The objective of ongoing research in our laboratory is the complete characterization of all constituents of epicuticular leaf waxes of W. nobilis. This will also allow comparisons of W. nobilis wax composition and morphology with that of other members of the Araucariaceae (Agathis and Araucaria).


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
LITERATURE CITED
 
The authors gratefully acknowledge the support by the Botanical Garden of the University of Würzburg (samples of Wollemia nobilis) and by J. Foundling, Bracknell, for providing the scanning electron micrographs. They thank Dr U. Hildebrandt, Dr M. Riedel and Dr G. Vogg for scientific comments and suggestions and O. Frank for skilful technical assistance. We are also indebted to Dr R. Jetter, University of British Columbia, for valuable comments on an earlier version of this manuscript. This work was supported by the Sonderforschungsbereich 567.


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 LITERATURE CITED
 

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