Chemistry and Crystal Growth of Plant Wax Tubules of Lotus (Nelumbo nucifera) and Nasturtium (Tropaeolum majus) Leaves on Technical Substrates Kerstin Koch,* Aarnoud Dommisse, and Wilhelm Barthlott
CRYSTAL GROWTH & DESIGN 2006 VOL. 6, NO. 11 2571-2578
Nees Institute for BiodiVersity of Plants, Rheinische Friedrich-Wilhelms UniVersity of Bonn, Meckenheimer Allee 170, D-53115 Bonn, Germany ReceiVed January 19, 2006; ReVised Manuscript ReceiVed September 7, 2006
W This paper contains enhanced objects available on the Internet at http://pubs.acs.org/crystal. ABSTRACT: Waxes consisting of hydrophobic and crystalline microstructures form multifunctional surfaces on a great number of plants. Scanning electron microscopy (SEM) and chemical analysis of extracted leaf waxes of lotus (Nelumbo nucifera Gaertn.) and nasturtium (Tropaeolum majus L.) showed that waxes of both species form small tubules and are composed of a mixture of aliphatic compounds, principally nonacosanol and nonacosanediols. Atomic force microscopy (AFM) and SEM were used to study the recrystallization of these wax tubules on technical surfaces. AFM studies provided consecutive images of the tubule formation process showing that precursor morphologies form a circular basis, which form tubules by continuous accumulation of new wax molecules. Recrystallization experiments showed wax crystal morphologies similar to those on plant surfaces, but great differences were found in tubules orientation and spatial distribution on the different substrates used. On silicon substrates, waxes preferably formed thick crusts, with randomly orientated tubules on top. On highly ordered pyrolytic graphite (HOPG) tubules predominantly grew vertically to the substrate in an ordered spatial distribution. Furthermore, on HOPG the recrystallized waxes decreased the substrate wettability with approximately 40° for lotus and 30° for nasturtium waxes. Introduction All primary surfaces of land plants are covered with a biopolymer referred to as a “cuticle”. On most cuticles, a mixture of hydrophobic compounds called “waxes” form threedimensional (3D) crystals.1,2 These epicuticular waxes are of central importance for the interaction of plants with their environment.3 With the establishment of analytical methods and high-resolution electron microscopy techniques in the 1970s, the morphology and chemistry of waxes have been studied intensively.4,5 Most waxes are derivatives of hydrocarbons, with substituted groups in terminal (e.g., primary alcohols, aldehydes) or nonterminal positions (e.g., β-diketones, secondary alcohols). Exceptions are polymeric aldehydes, and cyclic compounds such as flavonoids. Epicuticular waxes show a great morphological variability of 3D nano- and microprojections as platelets, rods, and tubules, and most of the 3D structures appear together with an underlying wax film.2,6-10 It has been understood that the diversity of wax micromorphology on plant surfaces results from self-assembly processes, based mainly on the chemical composition of the waxes. The most recent overviews of the classification and terminology of epicuticular waxes are given by Jeffree5 and Barthlott et al.2 The crystalline nature of plant waxes has been verified by X-ray powder diffraction (XRD) and electron diffraction (ED)1,11-14 and nuclear magnetic resonance (NMR) techniques.15 With an organic solvent (e.g., chloroform), waxes can be dissolved and removed from the leaves and recrystallized invitro on technical surfaces. Recrystallization of plant waxes led to a better understanding of the correlation between wax morphology and chemistry and is a suitable method for studying the self-assembly process of plant waxes. * To whom correspondence should be addressed. E-mail: koch@ uni-bonn.de.
Wax tubules can be distinguished in three chemical and morphological different groups.16 The first type contains high amounts of secondary alcohols, predominantly nonacosan-10ol and its homologues, the second type contains high amounts of alkanediols, and the third type of tubules contains high amounts of β-diketones. The recrystallization of a common wax type called “nonacosanol wax tubules”17,18 has been the emphasis of some earlier work,7,10,19,20 but there are still several unanswered basic questions, such as the mechanism of crystal growth. One hypothesis is that wax tubule formation results from a rolling in of platelet or ribbonlike precursors along their unattached edges.16 Twisted ribbons have been observed after recrystallization of tubular waxes19 and were termed as transitions between platelets and tubules. The combined occurrence of curved platelets with tubules on neighboring plant cells or even on one cell surface6,21,22 also led to the assumption that curved platelets are intermediate forms of tubules. This hypothesis of the tubule formation process is based on the work of Hallam,6,21 who discussed the tubule formation mechanism in general but did not differentiate between the types of tubules with different chemical compositions, of which the morphology differences are only in the tubule length.16 The hypothesis that curved platelets are intermediate forms of tubules is also strengthened by the observation of helical edges on the outer tubule wall, which have been interpreted as the edges of the fused platelets or ribbons.19 However, the platelet folding has never been observed, and recent findings of wax crystal growth23,24 point to a continuous growth of wax crystals by integrating new molecules at edges of the growing crystal. Therefore, an atomic force microscope (AFM) was used to study the dynamic processes of crystallization of the nonacosanol wax tubules. Waxes of lotus (N. nucifera Gaertn.) and nasturtium (T. majus L.) were chosen for this study because their leaves are densely covered with tubular wax crystals (Figure 1) containing the secondary alcohol nonacosan-10-ol
10.1021/cg060035w CCC: $33.50 © 2006 American Chemical Society Published on Web 10/17/2006
2572 Crystal Growth & Design, Vol. 6, No. 11, 2006
Figure 1. SEM images of nasturtium wax tubules on the upper leaf surface. The inset shows the wax tubules in detail.
and nonacosanediols as their main compounds.19,25,26 Scanning electron microscopy (SEM) was used for the micromorphological characterization of the final crystal shape and the crystal distribution on the substrates. Because the chemical composition of plant waxes shows great variations among plant species, as well as during organ ontogeny,27 the waxes used here were characterized via gas chromatography combined with mass spectrometry (GC-MS). Furthermore, the chemical composition of the lotus leaf wax was only known partially until now. Experimental Section Plant Material. Lotus (N. nucifera Gaertn.) and nasturtium (T. majus L.) plants were cultivated in the Botanical Gardens of the University of Bonn (Germany). Five leaves of lotus and approximately 150 leaves of nasturtium were harvested at the end of July 2003 for wax extraction. Wax Extraction. The freshly harvested leaves of lotus and nasturtium were dipped into chloroform (Applichem, Darmstadt, Germany) at room temperature. SEM of the immersed leaves showed that wax tubules on the leaves of both species dissolved within 2 s; thus, this time period was chosen for wax extraction to minimize possible contamination by waxes dissolved from the cuticle.28 The wax-chloroform solutions were filtered (filter paper grade 595; Schleicher and Schuell, Dassel, Germany) for removal of leaf contaminating particles from the extracted waxes. Solutions of 1.5 mg/ mL wax in chloroform were prepared. Chemical Analysis (GC). The extracted waxes were analyzed via capillary gas chromatography (HP 5890 series II, Avondale, USA) with on-column injection (30 m DB-1 i.d. 0.32 mm, film 0.2 µm; J&W Scientific, Folsom, California, USA) and a flame ionization detector (GC-FID, HP 5890 series II, Avondale, USA). The alcohols and carboxylic acids were transformed into the corresponding trimethylsilyl (TMS) derivatives by reaction with N,N-bis-trimethylsilyl-trifluoroacetamide (BSTFA; Macherey-Nagel, Du¨ren, Germany) in pyridine (Fluka Chemie, Buchs, Switzerland) for 45 min at 70 °C. One microliter of the samples was injected into the chromatograph. Quantification of the single compounds was done referring to an internal standard [20 µg of tetracosane (Fluka Chemie, Buchs, Switzerland)], which was added to the wax samples directly before GC analysis. Identification of the wax compounds was done using mass spectrometry (quadrupole mass selective detector HP 5971, Avondale, USA). All measurements were repeated twice with leaves of different plants and reported as mean values with their mean absolute deviation. Scanning Electron Microscopy (SEM). A LEO 440i scanning electron microscope (LEO, Oberkochen, Germany) was used to characterize the shape of the recrystallized waxes. Specimens (1.0 × 1.0 cm) were mounted on aluminum stubs with double-sided adhesive tape and coated with gold (approximately 25-30 nm) in a sputter coater (Balzers SCD040, Balzers, Liechtenstein). SEM examinations were
Koch et al. made at 15 kV. For SEM investigations, 10-µL droplets of the waxchloroform solutions, containing approximately 15 µg of wax, were placed on the substrates and specimens were stored in a desiccator. The droplets covered an area varying from 0.35 to 0.66 cm2, which resulted in a wax coverage of 23-43 µg/cm2. However, the wax coverage over the substrates was not homogeneous, and therefore local differences in wax coverage have to be taken care of. Over a period of 14 days, three specimens were investigated each day. The statistical analysis of spatial orientation of recrystallized waxes was made after 12 days and approximately 100 µm2 areas of five different specimens were analyzed. Atomic Force Microscopy (AFM). AFM measurements were carried out with a NanoScope IIIa (Digital Instruments, Mannheim, Germany) using tapping mode with aluminum coated silicon tappingmode tips (JPK-Instruments, Berlin, Germany). The laser beam intensity was reduced via an attenuation filter above the cantilever to diminish the thermal influence on the wax crystals. Temperature control was performed with a thermocouple. Temperatures of the specimen holders ranged from 22 °C, at the beginning of AFM investigations, to 26 °C 2 h later. Appropriate AFM conditions were a scan size of 3-20 µm, a scan rate of 0.5-2 lines/s with an image size of 256 × 256 pixel (experimental setup i) and 512 × 512 pixel (slower scan in experimental setup ii). A set point near the upper limit was chosen to minimize the interaction between tip and sample. For real time observations of the wax recrystallization by AFM, a 10-µL droplet of the wax-chloroform solution, containing approximately 15 µg of wax (see previous paragraph), was placed on the substrate, which was attached to a stainless steel disk with doublesided adhesive tape. AFM investigations started after evaporation of the chloroform. The first image acquisition started as soon as possible, approximately 6 min after application of the wax-chloroform solution. In the first mode (i) AFM images were consecutively taken of the same substrate area; thus, growth of the same crystals was observed over 3-4 h. These investigations have been repeated five times with each wax type. However, wax crystals could be affected by the scanning tip even in tapping mode and particularly at higher magnifications (scan size < 3 µm). Thus, a second mode (ii) was used for a control experiment. In this mode, each image was scanned at a different, yet adjacent area of the specimen to measure the size and distribution of the wax crystals with minimized scanning artifacts. Changes in crystal micromorphology were analyzed within a standard area of 6 × 6 µm over 5 h. Height and length measurements of single wax crystals in mode (i) were made with the program WSxM (Version 3.0; Nanotec Electronica, Madrid, Spain). The heights of several crystals in mode (ii) were measured using the image analysis tool “Bearing” of the AFM itself. Substrates. Freshly cleaved highly ordered pyrolytic graphite (HOPG, mosaic spread 3.5° ( 1.5°; Mateck, Ju¨lich, Germany) was used for studying the recrystallization processes since it has atomically flat surfaces well suited for high-resolution studies via AFM. Additionally, pieces of a silicon wafer (Si(100); Mateck, Ju¨lich, Germany) were used as substrates for wax recrystallization. The silicon wafer pieces were rinsed in ethanol at room temperature and in chloroform at 50 °C before applying the wax solutions. Wettability. The wettability of the HOPG substrate before and after covering it with wax crystals was measured via contact angle measurements (Dataphysics SCA 2.02, Filderstadt, Germany). Five microliters of demineralized water were automatically applied on top of the samples. and the contact angles were automatically determined using the Laplace-Young fitting algorithm. Each measurement was repeated six times.
Results Chemical Composition of Nasturtium and Lotus Waxes. The leaves of both investigated species were immersed in chloroform for 2 s, and the extracted waxes were analyzed by GC-FID and GC-MS. The chemical analysis (Table 1) showed that nasturtium wax is principally composed of the secondary alcohol nonacosan-10-ol [66.7% by weight (w %)]. Different nonacosanediols (8.4 w %) and alkanediols with other chain lengths (1.8 w %) were found as well. In total 93.8 w % of the detected peaks were identified, which include all major peaks
Crystal Growth of Plant Wax Tubules
Crystal Growth & Design, Vol. 6, No. 11, 2006 2573
Table 1. Gas Chromatographic Analysis of the Extracted Leaf Waxes of Nasturtium and Lotusa nasturtium (w %)
components Alkanes nonacosane
2.7 ( 0.6
Primary Alcohols 1.9 ( 0.3 0.2b 2.6 ( 0.6
octacosanol nonacosanol triacontanol nonacosan-10-ol triacontan-7-ol
lotus (w %)
Secondary Alcohols 66.7 ( 2.7
16.2 ( 1.1 2.4 ( 0.4
Alkanediols 1.3b 0.5b 3.1 ( 0.0 4.0 ( 0.2 1.3 ( 0.0
heptacosane-9,10-diol octacosane-9,10-diol nonacosane-3,10-diol nonacosane-4,10-diol nonacosane-5,10-diol nonacosane-10,13-diol hentriacontane-12,15-diol tritriacontane-9,10-diol Aliphatic Acids
1.5b 2.3b 0.6b
octadecanoic acid octacosanoic acid triacontanoic acid
18.6 ( 0.5 34.1 ( 1.9 12.0 ( 0.7 1.8 ( 0.0 0.7 ( 0.0 0.7 ( 0.0
Aldehydes octacosanal triacontanal
0.6b 0.6b
Esters of octacosanoic acid and decanol triacontanoic acid and decanol hexadecanoic acid and octacosanol
1.1b 0.9b 1.9b
not identified (
