Localization of the Transpiration Barrier in the Epi- and Intracuticular Waxes of Eight Plant Species: Water Transport Resistances Are Associated with Fatty Acyl Rather Than Alicyclic Components

Plant cuticular waxes play a crucial role in limiting nonstomatal water loss. The goal of this study was to localize the transpiration barrier within the layered structure of cuticles of eight selected plant species and to put its physiological function into context with the chemical composition of the intracuticular and epicuticular wax layers. Four plant species (Tetrastigma voinierianum, Oreopanax guatemalensis, Monstera deliciosa, and Schefflera elegantissima) contained only very-long-chain fatty acid (VLCFA) derivatives such as alcohols, alkyl esters, aldehydes, and alkanes in their waxes. Even though the epicuticular and intracuticular waxes of these species had very similar compositions, only the intracuticular wax was important for the transpiration barrier. In contrast, four other species (Citrus aurantium, Euonymus japonica, Clusia flava, and Garcinia spicata) had waxes containing VLCFA derivatives, together with high percentages of alicyclic compounds (triterpenoids, steroids, or tocopherols) largely restricted to the intracuticular wax layer. In these species, both the epicuticular and intracuticular waxes contributed equally to the cuticular transpiration barrier. We conclude that the cuticular transpiration barrier is primarily formed by the intracuticular wax but that the epicuticular wax layer may also contribute to it, depending on species-specific cuticle composition. The barrier is associated mainly with VLCFA derivatives and less (if at all) with alicyclic wax constituents. The sealing properties of the epicuticular and intracuticular layers were not correlated with other characteristics, such as the absolute wax amounts and thicknesses of these layers.The plant cuticle is one of the major adaptations of vascular plants for life in the atmospheric environment. Accordingly, the primary function of cuticles is to limit nonstomatal water loss and, thus, to protect plants against drought stress (Burghardt and Riederer, 2006). However, plant cuticles also play roles in minimizing the adhesion of dust, pollen, and spores (Barthlott and Neinhuis, 1997), protecting tissues from UV radiation (Krauss et al., 1997; Solovchenko and Merzlyak, 2003), mediating biotic interactions with microbes (Carver and Gurr, 2006; Leveau, 2006; Hansjakob et al., 2010, 2011; Reisberg et al., 2012) as well as insects (Eigenbrode and Espelie, 1995; Müller and Riederer, 2005), and preventing deleterious fusions between different plant organs (Tanaka and Machida, 2013).Cuticles are composite (nonbilayer) membranes consisting of an insoluble polymer matrix and solvent-soluble waxes. The polymer matrix (MX) is mainly made of the hydroxy fatty acid polyester cutin (Nawrath, 2006) and also contains polysaccharides and proteins (Heredia, 2003). In contrast, cuticular waxes are complex mixtures of aliphatic compounds derived from very-long-chain fatty acids (VLCFAs) with hydrocarbon chains of C₂₀ and more (Jetter et al., 2007). Wax quantities and compositions vary greatly between plant species and, in many cases, even between organs and developmental stages. Diverse VLCFA derivatives can be present, including free fatty acids, aldehydes, ketones, primary and secondary alcohols, alkanes, and alkyl esters. Besides, the cuticular waxes of many plant species also contain cyclic compounds such as triterpenoids and aromatics.In order to characterize the physiological function of cuticular waxes, methods have been developed for the isolation of astomatous cuticles and the measurement of transpiration rates under exactly controlled conditions, so that well-defined physical transport parameters such as permeances and resistances can be determined and compared across species and organs (Schönherr and Lendzian, 1981; Kerstiens, 1996; Riederer and Schreiber, 2001; Lendzian, 2006). With these methods, it was demonstrated that the cuticular water permeance increases by up to 3 orders of magnitude upon wax removal, thus showing the central role of waxes as a transpiration barrier (Schönherr, 1976). Permeances for water determined so far with astomatous isolated leaf cuticular membranes (CMs) or in situ leaf cuticles range over 2.5 orders of magnitude, from 3.63 × 10⁻⁷ m s⁻¹ (Vanilla planifolia) to 7.7 × 10⁻⁵ m s⁻¹ (Maianthemum bifolium; Riederer and Schreiber, 2001).The species-dependent differences of both wax composition and permeance led to a search for correlations between cuticle structure and function. If such a structure-function relationship could be established, then it would become possible to select or alter wax composition in order to improve cuticle performance in crop species (Kosma and Jenks, 2007). However, all attempts to understand cuticle permeance based on cuticle composition have failed so far: correlations between wax amounts and permeances could not be established, contrary to the common assumption that thicker wax layers must provide better protection against desiccation (Schreiber and Riederer, 1996; Riederer and Schreiber, 2001). Similarly, a correlation between wax quality (i.e. the relative portions of its constituents) and permeance could also not be established to date (Burghardt and Riederer, 2006). It is not clear how certain wax components contribute to the vital barrier function of the cuticle.Previous attempts to establish wax structure-function relationships may have failed because only bulk wax properties were studied and important effects of substructures were averaged out. However, distinct compartments of wax exist within the cuticle, most prominently as a layer of intracuticular wax embedded within the MX and a layer of epicuticular wax deposited on the outer surface of the polymer (Jeffree, 2006). Over the last years, methods have been developed that allow the selective removal of epicuticular wax by adhesive surface stripping, followed by equally selective extraction of intracuticular wax (Jetter et al., 2000; Jetter and Schäffer, 2001). Chemical analyses showed that, for most plant species investigated to date, both wax layers have distinct compositions (Buschhaus and Jetter, 2011). The most pronounced differences between the layers were found for the triterpenoids, which were localized predominantly (or even exclusively) in the intracuticular wax. These findings raised the possibility that the chemically distinct wax layers might also have distinct functions, leading back to the long-standing question of whether the water barrier function is exerted by the intracuticular and/or the epicuticular wax. There are only scant data to answer this question so far, mainly because methods allowing a distinction between epicuticular and intracuticular waxes were established only recently. Using these sampling techniques, it was recently found that, for leaves of Prunus laurocerasus, the epicuticular wax layer does not contribute to the transpiration barrier (Zeisler and Schreiber, 2016). In contrast, it had been reported that removal of the epicuticular wax layer from tomato (Solanum lycopersicum) fruit caused an approximately 2-fold increase in transpiration, suggesting that, in this species, the epicuticular layer constitutes an important part of the barrier (Vogg et al., 2004). Based on these conflicting reports, it is not clear to what extent the intracuticular or the epicuticular waxes contribute to the sealing function of the plant skin.The goal of this study was to localize the transpiration barrier within the cuticular membrane of selected plant species and to put the physiological function into context with the chemical composition of both the epicuticular and intracuticular wax layers. To this end, we selected eight species from which leaf cuticles could be isolated and methods for step-wise wax removal could be applied without damaging the cuticle. Preliminary studies had shown that the adaxial cuticles on leaves of Citrus aurantium (Rutaceae), Euonymus japonica (Celastraceae), Clusia flava (Clusiaceae), Garcinia spicata (Clusiaceae), Tetrastigma voinierianum (Vitaceae), Oreopanax guatemalensis (Araliaceae), Monstera deliciosa (Araceae), and Schefflera elegantissima (Araliaceae) were astomateous and showed wide chemical diversity. Therefore, these eight species were selected to address the following questions: (1) What are the amounts of epicuticular and intracuticular waxes? (2) Do compositional differences exist between the layers? (3) Where are the cuticular triterpenoids located? (4) How much do the epicuticular and intracuticular waxes contribute to the transpiration barrier? (5) Is the barrier associated with certain components of the intracuticular or epicuticular waxes?