Crystallographic studies of plant waxes Iris Meusel and Wilhelm Barthlott Botanisches Institut und Botanischer Garten der Universitat Bonn, Meckenheimer Allee 170, D-53115 Bonn, Germany Hartmut Kutzke and Bruno Barbier Mineralogisch-Petrologisches Institut der Universitat Bonn, Poppelsdorfer Schlofi, D-53115 Bonn, Germany (Received 1 September 1999; accepted 25 February 2000) Plant surfaces are mostly covered with microscopic layers of wax which exhibit characteristic morphologies, visible under high magnification. Waxes belonging to three different types were investigated. Powder data of seven natural and three recrystallised waxes as well as of two isolated compounds are presented. The mainly crystalline nature of the studied plant waxes is proved. The correlation between morphology, chemical composition, and powder patterns is discussed. © 2000 International Centre for Diffraction Data. [S0885-7156(00)00502-9] Key words: plant waxes I. INTRODUCTION Surfaces of leaves, stems, or fruits of most higher plants are covered with microscopic layers of lipids, so-called epi- cuticular waxes. Often they are visible as bluish-white, re- movable coatings, e.g., on grapes. An example of an extraor- dinarily massive wax layer is shown in Figure 1. Epicuticular waxes have been of great interest to many botanists for more than a century. Whereas in early publications macroscopic effects are described, e.g., light reflection or wettability (e.g., Sprengel, 1793), later works focus on microscopic studies of waxes. Nowadays, the micromorphology of epicuticular waxes can be investigated by high-resolution scanning elec- tron microscopy (SEM) showing three-dimensional images, as presented in Figure 2. A refined classification and termi- nology of epicuticular waxes based on high-resolution SEM analysis of at least 13 000 species, representing all major groups of seed plants, has been published by Barthlott et al. (1998). In total, 23 wax types were described: ubiquitous thin, unstructured wax films as well as distinct wax projec- tions ("wax projections" in botanical context means three- dimensional, microscopic structures of plant waxes). In the present study three wax types are investigated by X-ray dif- fraction. Schematic drawings are shown in Figure 3. The basic morphology of these waxes, apart from a few exceptions, is not influenced by the environment (Cutler and Brandham, 1977); therefore, it is a useful characteristic in the systematics of flowering plants (Barthlott, 1993). Moreover, wax projections produce a microscopically rough surface that is hydrophobic. This surface has important adaptive functions, including highly reduced wettability and great anti-adhesivity (survey in Barthlott, 1990). In addition, con- taminating particles (e.g., dust, pathogens) are carried away by rain drops, leading to a clean surface, which is known as the "lotus effect." The application of these properties to artificial surfaces may be important for the production of uncontaminable varnishes of cars and other manufactured surfaces. Chemically, plant waxes are composed of a large num- ber of different compounds, mostly long-chain aliphatics, which can be further classified according to their structure, substitution, and the distribution of their homologues. Cyclic compounds, however, may sometimes predominate. The chemistry of surface waxes has been extensively reviewed (Baker, 1982; Walton, 1990; Bianchi, 1995; Riederer and Markstadter, 1996), but it is noted that nothing is known about the chemical composition of isolated wax projections. Beginning in the 1800s, a close relationship between the particular shape and the chemical composition of epicuticu- lar waxes was assumed (de Bary, 1871). Nowadays it is com- monly accepted that the wax micromorphology chiefly de- pends on the chemical composition (survey in Jeffree, 1986). Usually, dominant wax components are considered to deter- mine the individual ultrastructure of waxes, through self- assembly. In some cases, especially for wax tubules, this close correlation can be demonstrated by recrystallisation of the total wax as well as isolated single components. Such recrystallised projections show the same form and a similar size as the corresponding projections on plant surfaces (Jetter and Riederer 1994, 1995; Meusel et al, 1999). As early as 1867, Uloth hypothesized the crystalline na- ture of plant waxes. Further investigations confirmed his sug- gestion: de Bary (1871) recrystallised some plant waxes and Wiesner (1876) observed the birefringence of several plant waxes. Some of the first X-ray investigations of plant waxes Figure 1. Eucalyptus macrophylla, operculum (lower part of the flower) covered with a grey-white massive wax layer. 123 Powder Diffraction 15 (2), June 2000 0885-7156/2000/15(2)/123/7/$6.00 © 2000 JCPDS-ICDD 123