INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Leaves of higher plants are covered by a cuticle, i.e. an extracellular membrane consisting of a polymeric cutin matrix and soluble cuticular waxes. A specific portion of the waxes is embedded within the polymer framework (Holloway, 1982) and should be designated as "intracuticular waxes" (Walton, 1990). It is widely accepted that all plant cuticles also carry a thin film of "epicuticular waxes" on the surface of their cutin matrix (Baker, 1982). In addition, on particular plant species and organs the surface waxes can form microscopic aggregates, "epicuticular wax crystals," protruding from the wax film (Barthlott et al., 1998).

Due to its position at the interface between the plant and its atmospheric environment, the cuticle performs multiple physiological and ecological roles. To be more specific, some of the most important properties of the cuticle must be determined by epicuticular wax films because they are located at its very surface. On one hand, there is circumstantial evidence that the most effective part of the cuticular transpiration barrier resides in waxes at or near the cuticle surface (Schönherr, 1976; Schönherr and Riederer, 1988). On the other hand, this outermost cuticular layer must necessarily be relevant for host recognition by fungi as well as insects. It has been shown that many pathogens and herbivores probe the chemical nature of their substrate (Schoonhoven et al., 1998). Wax constituents act as allelochemicals by influencing fungal development (Carver et al., 1990; Podila et al., 1993; Flaishman et al., 1995) and/or insect behavior (Städler, 1986; van Loon et al., 1992). To further understand the molecular mechanisms determining these essential functions of plant surfaces, epicuticular wax compositions must be documented with adequate spatial and temporal resolution.

In the past, surface extraction with organic solvents was routinely employed to probe cuticular waxes for chemical analysis (Holloway, 1984). Because solvent molecules release both intra- and epicuticular waxes together, only the overall composition of the complete wax mixture could be assessed (Walton, 1990). This bulk wax composition was usually assumed to reflect the surface composition, ignoring possible differences between intra- and epicuticular waxes. Meanwhile, some evidence for chemical differences between intra- and epicuticular waxes has emerged. First, comparative chemical and morphological investigations showed that special wax constituents form the epicuticular crystals on diverse plant surfaces (Jeffree et al., 1975; Gülz et al., 1992; Jetter and Riederer, 1994; Jetter and Riederer, 1999; Meusel et al., 1999; Markstädter et al., 2000). In these cases individual constituents are accumulated at the very plant surface (Jeffree et al., 1975; Baker, 1982), probably by diffusion and spontaneous phase separation of compounds. Second, for those plant surfaces covered by surface wax films without epicuticular crystals, a distinction between intra- and epicuticular constituents was attempted by either mechanically stripping (Haas and Rentschler, 1984) or chemically washing (Silva Fernandes et al., 1964; Holloway, 1974; Baker and Procopiou, 1975; Baker et al., 1975; Svenningsson, 1988; Garrec et al., 1995) the film off the plant surface. None of these methods had sufficient selectivity to unambigously localize individual compounds in either cuticular sublayer. However, in all cases transverse gradients in the percentages of individual compounds suggested chemical differences between intracuticular waxes and the surface film. Taken together, all these studies clearly indicated that surface composition cannot be extrapolated from extractive bulk wax mixtures.

First methods were devised only recently allowing the selective preparation of epicuticular wax films and hence the direct analysis of surface compositions (Ensikat et al., 2000; Jetter et al., 2000). For Prunus laurocerasus leaves, a layered arrangement of wax fractions was revealed, with aliphatic constituents concentrated in the epicuticular film and pentacyclic triterpenoids located exclusively in the intracuticular compartment. In these experiments, transverse gradients within the cuticular components were quantified for the first time. However, because wax preparation included freeze-thaw cycles of the leaf tissue the methods could not be applied in vivo to study the dynamics of cuticular wax layers. Crucial aspects of cuticle formation, especially the mechanisms and regulation of surface wax arrangement, hence remained unknown. As a consequence, we now initiated in vivo investigations into the seasonal development of epicuticular waxes of P. laurocerasus. The main objective of the present work was to assess: (a) the rates for the accumulation of individual compound classes at the leaf surface, (b) the processes leading to segregation of intra- and epicuticular wax fractions, and (c) the mechanisms explaining the net loss of epicuticular wax constituents. To investigate all these aspects of cuticle dynamics, new procedures had to be developed employing adhesives to selectively probe epicuticular waxes in situ.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

The first objective of the present investigation was to develop methods for selectively probing epicuticular wax films in situ. It had previously been shown that adhesives can be employed to mechanically remove surface waxes for chemical analysis (Jetter et al., 2000). The most selective method employing frozen glycerol as cryo-adhesive was used to generate reference data on the epicuticular wax composition of adult P. laurocerasus leaves. This yielded a characteristic pattern of C₂₅ through C₃₅ alkanes, C₂₁ through C₃₆ alcohols, C₂₃ through C₃₆ aldehydes, C₂₀ through C₃₄ fatty acids, and C₂₂ through C₃₆ acetates (Fig. 1A). Diverse other adhesive materials were now tested as alternatives to remove surface compounds from the leaves.

View larger version (43K): [in this window] [in a new window]   Figure 1.   Composition of wax mixtures removed from adaxial surfaces of mature P. laurocerasus leaves by adhesive treatment or solvent extraction. Yields of individual compound classes (µg cm2) are given as mean values (n = 6) with SD. A, Surface waxes were sampled in a single treatment employing frozen glycerol as a cryo-adhesive or in three consecutive treatments with an adhesive polymer film formed by an aqueous solution of gum arabic. B, Comparison between wax mixtures removed by repeated gum arabic treatments, released by successive chloroform extraction of the same surface, or by extraction of the native surface (without prior mechanical treatment).

Various glues yielded wax mixtures similar to those of cryo-adhesive treatments, but the overall amount, pattern of individual compound classes, and chain length distributions were best reproduced with gum arabic. Repeated treatments of a given surface with this polymer released mixtures of similar composition (Fig. 1A) but with drastically decreasing yields. In none of the mechanically prepared wax samples triterpenoids could be detected. Only when the gum arabic-treated leaf discs subsequently were extracted with chloroform were two triterpenoids, ursolic and oleanolic acid, released (Fig. 1B). Besides, small amounts of the aliphatic compound classes previously identified in gum arabic preparations could be detected in the solvent extracts. The total amounts both of individual constituents and of compound classes removed by three successive gum arabic treatments plus subsequent chloroform extraction were equal to their yields in a single extraction of authentic surfaces (Fig. 1B).

Further experiments showed that gum arabic could be left for up to 10 d on P. laurocerasus leaves without causing visible damage. The polymer could also be applied to and removed from immature leaves without disturbing their development. Hence, a detailed investigation using gum arabic to study the seasonal development of adaxial leaf surfaces was initiated. The polymer was employed in situ to remove the epicuticular wax film from the entire surface of leaves in the earliest accessible stage of development. This was first possible 3 d after bud break and thus would define the starting point in the time course. The initially removed epicuticular waxes contained high percentages of acetates with small admixtures of alkanes, alcohols, and fatty acids (Table I). Homolog distributions ranged from C₂₀ to C₃₆ with a predominance of chain lengths C₂₄ and C₂₆. The total yield of removed epicuticular compounds corresponded to an original coverage of 4.0 ± 0.94 µg cm².

View this table: [in this window] [in a new window]   Table I.   Composition of epicuticular waxes on adaxial surfaces of P. laurocerasus leaves 3 d after bud break Mean values (n = 20) and SD are given for the homologous composition (% of the fraction) and the coverage (µg/cm2) of compound classes.

Leaf development was monitored for 57 d after the first treatment with gum arabic. During the first 10 d leaf surface areas increased linearly with continuously growing variability (Fig. 2A, dotted line). In the same time interval, a steady decrease in the density of epidermal cells per unit area was observed (Fig. 2B) and the total number of cells per leaf (calculated from data in Fig. 2, A and B) remained constant at approximately 3.6 10⁶ (Fig. 2C). This first phase of the time course thus was characterized by expansive growth of epidermal cells. During the ensuing time interval (d 13-57) the leaf surface area, epidermal cell density and total cell numbers remained approximately unchanged (Fig. 2). In the developmental investigation of surface waxes, the chemical data from all time points had to be compared and therefore were normalized using standard leaf areas. These were calculated by performing a linear regression for the phase of cell expansion (A_d = 0 = 5.8 cm²; A = 2.2 cm² d¹) and by averaging the final area of all individual leaves (A = 29.1 cm²; Fig. 2A, bold lines). This procedure helped to eliminate small fluctuations in some of the values, especially at d 30 and 43, that were caused by overrepresentation of certain leaf batches in respective samples. Because the six individual leaves sampled and analyzed at d 30 and 43 mostly belonged to batch E (latest bud break), they had relatively small final areas (Fig. 2A) and epidermal cell numbers (Fig. 2C). For all individual leaves in the present investigation, a strict correlation was found between the date of bud break, the time before they reached full size, and their final surface area.

View larger version (24K): [in this window] [in a new window]   Figure 2.   Size development of P. laurocerasus leaves. Averages and SD (n = 6) for the surface areas (A), the epidermal cell densities (B), and the calculated (approximate) epidermal cell numbers (C) are given for all the leaves sampled in the present investigation. Straight lines in A show the average area values used for normalizing wax coverage data.

Development of epicuticular wax coverage and composition during a period of 57 d after the first wax removal was studied. For this purpose, at each sampling point six leaves were selected and their surface waxes were probed with a second gum arabic treatment and analyzed. Total yields of epicuticular waxes per unit area rose steeply from 0 to approximatley 3 µg cm² in the 5 d after the first removal. In the ensuing time interval to d 57, a smooth overall increase to 6 µg cm² could be seen. A drastic rise and loss of coverages around d 23 transiently was observed before moderate increase was resumed. Because the leaf areas remained constant over most of the investigated period, the epicuticular wax amounts per leaf were very similiar to the development of coverages per unit area. Hence, changes in wax composition can be bilanced on a whole-leaf basis without loss of information.

In the development of adaxial surface waxes of P. laurocerasus leaves, different phases could be distinguished according to the amounts of individual compound classes present (Fig. 3). First, a steady increase in the absolute quantities of acetates during 8 d after the initial wax removal resulted in a strong predominance of this compound class. In the course of the ensuing development, the total amounts of acetates on the adaxial surface steadily declined. In a converse manner, the quantities of alcohols steadily increased between d 8 and 23, at first compensating and then exceeding the loss of acetates. Total leaf coverages consequently leveled off beween d 8 and 13 before they reached a maximum at d 23. Due to losses of the predominating alcohols, the total wax amounts were again diminished in the following phase. Only after d 30 an increase in the absolute quantities of alkanes could compensate for further deprivation of both alcohols and acetates, and the epicuticular wax mixture was then dominated by alkanes. The aldehydes and alkyl esters, although at lower amounts, showed developments similar to the alkanes, whereas the time course of fatty acid quantities paralleled that of the alcohols.

View larger version (62K): [in this window] [in a new window]   Figure 3.   Development of epicuticular waxes on adaxial surfaces of P. laurocerasus after an initial treatment with gum arabic. A single treatment with gum arabic was employed to selectively remove surface waxes. Total amounts (average ± SD [n = 6]; µg leaf1) of epicuticular waxes (gray area) and their composition were determined by gas chromatographic (GC) analysis.

It should be noted that an epicuticular wax layer that was chemically indistinguishable from the original film (Table I) accumulated within 2 d after the initial wax removal (Fig. 3). This early increase of waxes could either reflect the normal development or a restoration of the disturbed surface. To distinguish between these possibilities, the epicuticular film was investigated on control leaves without initial wax removal. Gum arabic treatment at d 5 of the present time frame yielded 54 µg cm² of wax containing 91% (w/w) alcohol acetates. In a similar manner, wax amounts and composition at d 8, 10, and 57 were identical to those on leaves from which waxes had originally been removed (data not shown). This shows that the normal ontogenetic development of epicuticular waxes had been resumed within 5 d after the initial wax removal. Hence, in Figure 3 chemical data for d 1 and 3 reflect epicuticular wax regeneration, whereas those for d  5 characterize the normal surface ontogeny.

The chain length distribution of compounds within the major fractions of P. laurocerasus waxes changed drastically during development (Fig. 4). Acetates, alcohols, and fatty acids showed a clear predominance of the C₂₄ homologs with admixtures of C₂₆ compounds at d 8. In contrast, after d 23 the acetate fraction had a homolog pattern centered around C₃₂. The chain length distribution of alcohols and fatty acids changed more gradually and reached a characteristic new pattern at d 57. In both compound classes then, C₃₀ compounds were slightly predominating. The homolog distribution of alkanes was only slightly altered in the same time interval, showing a steady shift from compounds with 29 carbon atoms to homologs with 31 carbons over time (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 4.   Chain length patterns of major aliphatic compound classes in epicuticular waxes from adaxial P. laurocerasus leaf surfaces. Homolog distributions (average ± SD [n = 6]; %) in the acetate (A), in the alcohol (B), and in the fatty acid fractions (C) were calculated from individual compound yields in epicuticular wax mixtures sampled with gum arabic.

Because the homolog patterns of acetates, alcohols, and fatty acids changed most drastically during leaf development, the complete time course of these compounds has to be further inspected. To this end, the amounts of even-numbered homologs centered around C₂₄ and C₃₂, respectively, can be compared because they showed most pronounced changes in the investigated time interval (compare with Fig. 4). Acetates with relatively short chain lengths were accumulated only before d 8 and then quickly lost (Fig. 5A). In contrast, higher acetate homologs were slowly increasing during the first 23 d of development, then slightly decreasing and leveling off at approximately 10 µg leaf¹. The latter time course was exactly paralleled by both shorter and longer homologs of alcohols (Fig. 5B). Only the fatty acids with relatively short chain lengths showed a development similar to the corresponding alcohols, whereas the longer fatty acid homologs were steadily increasing over the whole time course (Fig. 5C).

View larger version (30K): [in this window] [in a new window]   Figure 5.   Time course of selected homologs of major compound classes in epicuticular waxes from adaxial P. laurocerasus leaf surfaces. Coverages (average ± SD [n = 6]; µg leaf1) of individual homologs were determined in epicuticular wax mixtures prepared with gum arabic. Added amounts for acetate (A), alcohol (B), and fatty acid homologs (C) with relatively short- (or long-) chain lengths are plotted as a function of leaf development.

The mechanical removal of superficial wax films from immature leaves of P. laurocerasus was visualized by scanning electron microscopy (SEM). Two days after bud break, the adaxial surface of leaves was made up by undulated epidermal cells with irregular polygonal outlines (Fig. 6A). Anticlinal cell boundaries were marked by sharp grooves, whereas periclinal surfaces were structured by smooth ridges and sporadic granules. Indentations delineating cell boundaries were inversely replicated on the gum arabic film removed from respective surfaces (Fig. 6B). Because substantial quantities of waxes were detected in authentic gum arabic preparations, it can be assumed that the polymer was coated with a wax film. The visible surface thus represents the boundary layer between the mechanically removed waxes and the remainder of the leaf cuticle. On very young leaves, the treatment with gum arabic did not leave microscopically visible traces. However, on leaves more than 10 d old a rough line bordering the polymer-treated area could be found (Fig. 6, C and D). On the native surface of these leaves granular structures had accumulated that were removed by gum arabic together with the continuously underlying wax film. The largely unbroken film with interspersed granules alternatively could be deposited on glass using cryo-adhesives as previously described (Fig. 6, E and F). The film thickness increased markedly from approximately 25 nm at d 10 of the time course to 100 nm at d 57. 

View larger version (197K): [in this window] [in a new window]   Figure 6.   Scanning electron micrographs of adaxial P. laurocerasus leaf surfaces. A, Untreated control 2 d after bud break. B, Gum arabic replica of the same developmental phase viewed from the side of the polymer that had been in contact with the leaf. C and D, Surface of leaves 15 d after bud break: Granular structures mark the native surface (upper half) whereas areas treated with gum arabic are smooth (lower half). Both zones are delineated by the rim of the epicuticular film (white arrows). E and F, Film of epicuticular waxes from leaves 15 d after bud break (right side) transferred onto glass (left side) using frozen water.

In parallel to the developmental studies described above, a selection of P. laurocerasus leaves was probed with gum arabic without initial disruption of the wax film (defining d 0 in the previous studies). Leaves were sampled at developmental stages corresponding to d 5, 8, 10, and 57 in the original time frame (see above). After removal of epicuticular waxes respective leaves were harvested and adaxial surfaces were extracted with chloroform. The resulting mixtures of intracuticular waxes were dominated by the triterpenoids oleanolic and ursolic acid (Fig. 7). Their coverages increased drastically before d 8 and then slowly decreased from 900 to 600 µg leaf¹. The aliphatic portions of the intracuticular mixtures were dominated by acetates at d 5, alcohols between d 8 and 10, and alkanes thereafter. In the alcohol fraction, homologs with chain lengths  C₂₆ prevailed at all sampling points. In contrast, the acetates consisted of relatively short chain lengths only at d 5, whereas chain lengths  C₂₈ predominated after d 8. 

View larger version (36K): [in this window] [in a new window]   Figure 7.   Composition of intracuticular waxes from adaxial sides of P. laurocerasus leaves. Coverages (average [n = 2]; µg leaf1) and, for alcohols and alkyl acetates, relative portions of homologs C26 (lower part of bars) and homologs C28 (upper part of bars) are given.

In a final experiment, the epicuticular wax film was removed with frozen water from several developing leaves (d 3 in the investigated time frame, corresponding to d 6 after bud break) and transferred to glass supports. Individual preparations were stored for 15 d directly at the mother plant (outdoors), whereas others were kept in the greenhouse. After this time, their composition was determined and compared with that of the initial mixture and the epicuticular wax film that meanwhile had developed on the leaf surface. In vitro, both the relative composition and the coverage of all aliphatic film constituents, quantified as compound classes and as homolog patterns therein, were unaltered (Fig. 8), whereas the in vivo control confirmed the drastic decrease of acetates and the accumulation of alcohols (see above).

View larger version (27K): [in this window] [in a new window]   Figure 8.   In vitro and in vivo development of epicuticular wax films from adaxial sides of P. laurocerasus leaves starting 5 d after budbreak. Wax films were transferred onto glass and stored for 15 d. Coverages (average ± SD [n = 5]; µg cm2) of individual compound classes were then compared both with the initial composition and to the epicuticular film that had developed on control leaves.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

For the developmental investigations of the P. laurocerasus leaf surface, a method had to be devised that would allow the nondestructive removal and chemical analysis of waxes from living plant organs. For this purpose, an adhesive film should be formed on the leaf surface and then be removed together with adhering waxes without disturbing the tissue. The adhesives that had previously been employed in similar studies either could not sufficiently discriminate between intracuticular and epicuticular material (Haas and Rentschler, 1984) or included freeze-thaw cycles that impeded an in vivo application (Jetter et al., 2000). We now found that both problems could be overcome using gum arabic as an adhesive. Parallel samples that were prepared from comparable P. laurocerasus leaves showed that coverages (µg cm²) of individual compounds could be determined with high reproducibility.

It had been reported that wax removal with cryo-adhesives is highly selective for epicuticular material because it relies entirely on the mechanical interaction between adhesive and wax films (Jetter et al., 2000). For authentical material, the wax mixtures prepared with gum arabic and cryo-adhesives were nearly identical in total amounts and composition. Therefore, both methods show equally high selectivity for epicuticular waxes. This was further verified by experiments in which the same leaf surface was subjected to multiple gum arabic treatments and a final extraction. The sharply declining yields of repeated mechanical wax removal showed that a physically resistant boundary had been reached. Beyond this interface, more material could only be released by solvent extraction. As for the cryo-adhesive technique, the physical boundary thus characterized must be identical with the outer limits of the cutin matrix. Hence, gum arabic treatment yielded exclusively epicuticular material, as defined by the location on the outside of the cutin matrix, whereas the consecutive extraction released the remaining intracuticular material. The triterpenoid acids serve as intracuticular markers being released neither by cryo-adhesives nor by gum arabic treatment. Previous attempts to selectively prepare epicuticular waxes by differential extraction (Silva Fernandes et al., 1964; Holloway, 1974; Baker and Procopiou, 1975; Baker et al., 1975; Svenningsson, 1988; Garrec et al., 1995) suffered from cross-contamination with intracuticular waxes and could hence only qualitatively describe surface compositions. With the new adhesive methods, the outermost layers of plant cuticles now can be quantitatively analyzed.

In the course of the developmental investigation of adaxial P. laurocerasus leaf surfaces, the mechanically removed epicuticular wax films were visualized by SEM. As judged after transfer to glass surfaces, the epicuticular films on leaves of all developmental stages were contiguous. After leaf expansion had ceased, the film thickness increased steadily, corresponding to the observed increases in epicuticular wax loads. For wax coverages of 3 and 6 µg cm² (corresponding to d 10 and 57 in the investigated time frame), and based on density values of very-long-chain aliphatic compounds of 0.8 to 1.0 10⁶ g m³ (Le Roux, 1969), a film thickness of 30 to 40 nm and 60 to 75 nm can be expected, respectively. These chemical estimates and the SEM representation of the epicuticular film thickness at various developmental stages were hence in good accordance. For fully developed P. laurocerasus leaves (more than 1 year old), an adaxial wax film thickness of 130 to 160 nm had been reported (Jetter et al., 2000) and the present data document a steady net accumulation of material slowly forming the mature surface. Assuming that wax molecules are all cis-configured (Small, 1984) and oriented perpendicular to the leaf surface (Sitte and Rennier, 1963) the epicuticular film on 10-d-old leaves was approximately 10 molecules thick. During the following 50 d its thickness had increased to approximately 20 molecular layers, whereas mature leaves showed a film thickness corresponding to approximately 35 to 45 layers (Jetter et al., 2000).

The epicuticular wax film was removed initially to create a controlled starting situation. Original wax amounts and compositions were restored after approximately 3 d and further development of epicuticular waxes was not disturbed by the initial removal, as control experiments with single probing (starting at d 5 in the present time frame) showed. This demonstrates that epicuticular waxes on leaves of P. laurocerasus can be regenerated at least in the early stages of leaf development. In accordance with this result, surfaces of diverse Eucalyptus and Brassica species had previously been shown to reform epicuticular waxes especially on young leaves (Hallam, 1970; Percy and Baker, 1987; Wolter et al., 1988; Wirthensohn and Sedgley, 1996). As in these studies, the regenerated waxes were only monitored by SEM and/or the originally removed wax layer was not analyzed, the rates of wax restoration could not be quantified. The regenerative capacities of plant surfaces can now be quantitatively studied as a function of developmental stages. The epicuticular plasticity of P. laurocerasus and other model species is currently being investigated with the methods developed here.

Earlier reports on epicuticular waxes of mature P. laurocerasus leaves were confirmed in the present investigation, demonstrating that both the absolute amounts and the relative composition of surface compounds are invariable after leaf development is completed (Stammitti et al., 1996; Jetter et al., 2000). In contrast, drastic changes occurred during organogenesis: Various compound classes, most prominently acetates and alcohols, sequentially appeared and were accumulated to high percentages at the plant surface. It is of special interest that multiple, distinct phases with extensive wax turnover occurred within few days. Only moderate shifts, either in the portions of compound classes, their coverages, or their chain length distributions, had been reported for waxes on other plant species (Tulloch, 1973; Stocker and Wanner, 1975; Baker et al., 1982; Prasad and Gülz, 1990; Gülz et al., 1991; Maier and Post-Beittenmiller, 1998; Rhee et al., 1998). These reports could not specify where respective alterations occurred within the cuticle because mixtures of epi- and intracuticular waxes had been analyzed. The present findings confirmed the general trend for longer chain homologs to accumulate in later developmental stages (Haas, 1977; Atkin and Hamilton, 1982; Jenks et al., 1996).

This is the first report on changes locally occurring in the epicuticular wax film. As a consequence, the question arises whether distinctive phases in epicuticular wax development are due to an ontogenetic program or merely reflect differences in the flux of biosynthetic products to the surface. First, intracuticular waxes contained high levels of triterpenoids throughout leaf development. The newly appearing aliphatic compounds hence had to move to the surface across a preformed intracuticular compartment. Therefore, it can be excluded that the distinct epi- and intracuticular regions were generated by sequential deposition of contiguous layers. Second, the steady accumulation of aliphatics in the epicuticular film, as monitored by changes in the amounts of compound classes and chain length distributions, was in all cases preceded by respective changes in the intracuticular compartment. This is further evidence for the separate movement of individual compounds toward the surface. In conclusion, phase separation likely occurs within the bulk cuticular waxes and holds triterpenoids in the intracuticular compartment, whereas aliphatics are partitioned between both layers.

Individual compound classes and homologs accumulate in the epicuticular compartment of P. laurocerasus leaves within a few days. Biosynthetic products must consequently move toward the plant surface with rates in the order of nanograms per centimeter² an hour, in accordance with reports for other plant species (Baker and Hunt, 1981). If both phase separation and transport (within the cuticle) are spontaneous then the resulting fluxes should be limited by diffusion rates. According to Schreiber and Schönherr (1993), the mobility of aliphatic molecules within the wax mixture will depend largely on their chain length, but not on their functional groups. Acetate and alcohol homologs centered around C₂₄ should have diffusion coefficients of approximately 10²⁰ m² s¹. Model calculations show that even with this small molecular mobility an adequate flux of material will spontaneously arise, if the compounds are delivered to the innermost parts of the cuticle in micromolar concentrations (Riederer and Schreiber, 1995). Because these concentrations are within reasonable limits, the flux rates of aliphatics to the leaf surface of P. laurocerasus can be sufficiently explained by diffusion.

The sequential appearance of diverse aliphatic compound classes at the plant surface conversely cannot be rationalized with differences in their diffusion rates. Based on their largely matching chain length distributions these compounds, e.g. the acetates and alcohols in respective phases of leaf development, would be expected to diffuse with similar rates (Schreiber and Schönherr, 1993). Instead of differential diffusion, the timed accumulation of epicuticular constituents therefore probably reflects the ontogenetic regulation of wax biosynthesis (or apoplastic export). Therefore, it will be interesting to compare future results on either the formation or the export of wax constituents with the present details on the temporal and spatial distribution of P. laurocerasus waxes.

Generally accepted models for the formation and structure of the plant cuticle were assuming that cuticular waxes are coordinately accumulated (Baker, 1982). In accordance, values for relative amounts (%) or coverages (µg cm²) of individual constituents may decrease over time when other compounds are added or when leaves expand, respectively. However, losses of individual homologs or whole compound classes on a whole-leaf basis (µg leaf¹) had been reported only scarcely (Markstädter, 1994; Riederer and Markstädter, 1996; Hauke and Schreiber, 1998). In P. laurocerasus epicuticular waxes, both acetates and alcohols were found to decrease independently by almost 4 µg leaf¹ d¹ during special periods in leaf development. Three alternative mechanisms could explain this loss of surface material: (a) The compounds could be converted in situ into other wax constituents, (b) they could be lost to the atmosphere by evaporation or erosion, or (c) they could be transported back to inner parts of the cuticle, the epidermal cell wall, or the protoplast, where they would be metabolized or transformed into other wax compounds.

The marked decrease of acetates in P. laurocerasus epicuticular wax coincides with an increase of alcohols with identical chain lengths. Therefore, the latter might be considered as in situ conversion products formed by acetate hydrolysis. This hypothesis was tested in an experiment using the methods developed here: Epicuticular wax films were isolated and exposed to natural climatic conditions. In vitro the aliphatic film constituents were unaltered, whereas the in vivo control confirmed the drastic changes detailed above. Because the wax composition is not affected when only the epicuticular film is exposed to its natural atmospheric environment, a spontaneous conversion of acetates into alcohols within the epicuticular waxes can be ruled out. This experiment also shows that neither evaporation nor erosion can account for the loss of acetates from the epicuticular film. Instead, the intracuticular compartment and/or the epidermis must be involved in the processes. Present evidence consequently favors the third mechanism, transport of epicuticular compounds back into the tissue followed by further conversion, to explain the loss of epicuticular compound classes during P. laurocerasus wax development. Labeling studies can be employed to further study the transport phenomena described in the present paper. With the techniques developed here, the leaf surface can be manipulated to perform adequate pulse-chase experiments.

Coverages of the intracuticular triterpenoids of P. laurocerasus leaves were diminishing with even higher rates than epicuticular compounds. Similar effects had previously been reported for triterpenoid acids in leaf waxes of other Rosaceae, namely Malus domestica (Hellmann and Stösser, 1992), Malus hupehensis, and Prunus cerasus (Baker and Hunt, 1981) as well as Prunus persica (Baker et al., 1979). Although epi- and intracuticular waxes were not differentiated in these studies, it seems likely that, similar to the closely related species P. laurocerasus, the triterpenoids are localized in inner parts of the cuticle. It has been surmised that the yields of superficial solvent extraction might not reflect the loss of compounds but instead the changes in their intracuticular environment (Jetter et al., 2000). The slowly progressing polymerization of the cutin matrix (Croteau and Kolattukudy, 1974; Schmidt and Schönherr, 1982; Kolattukudy, 1984; Khan and Marron, 1988; Riederer and Schönherr, 1988) could gradually restrict access for solvent molecules and would hamper extraction. The triterpenoid acids alternatively could react with functional groups on the cutin matrix, binding them to the polymer and hence rendering them insoluble. Similar processes have been reported for aromatic acids (Basford, 1991) and fatty acids (Hauke and Schreiber, 1998). Although these mechanisms could explain the reduced yields of intracuticular triterpenoid acids from older leaves, they cannot account for the losses of epicuticular acetates and alcohols. Hence, two different mechanisms, translocation of epicuticular waxes and changes in the intracuticular structure, might lead to the apparent loss of compounds from cuticular compartments.

The present data set bears implications related not only to cuticle structure and formation but also to the ecological and physiological properties of plant surfaces. The developmental changes in P. laurocerasus leaf wax composition require a precise regulation of wax biosynthesis and an investment of energy and material. Therefore, it seems very likely that specific functions are correlated with the different compound classes dominating the surface composition in distinct developmental phases. Because the accumulation of acetates coincides with the period of fastest leaf expansion, this compound class might play a special role in sealing the still thin, rapidly expanding cuticle (Schönherr, 1976; Schönherr and Riederer, 1988). On the other hand, the transient amassing of alcohols in the epicuticular film brings about that the surface composition of the developing leaf completely changes twice in short intervals. This might help to divert herbivorous insects that use either wax acetates or alcohols or alkanes as host surface markers by shortening the time windows in which the animals can recognize their host (Städler, 1986; Carver et al., 1990; van Loon et al., 1992; Podila et al., 1993; Flaishman et al., 1995). Our new methods allow to analyze and manipulate surface composition in situ. Hence, both the physiological and the ecological properties of the epicuticular wax film at different developmental stages can now be investigated.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant Materials

Small trees of Prunus laurocerasus were continuously grown in pots in the greenhouses of the Botanical Garden of the University of Würzburg. For method development, mature leaves were harvested randomly from three individual plants in fall and winter and were pooled for wax removal and analysis. Leaf discs with 20-mm diameter were cut from these leaves and used to compare the reproducibility and selectivity of mechanical wax removal using either cryo-adhesives or gum arabic.

A single tree was moved into the garden in the springtime 2 weeks before developmental studies of epicuticular waxes began. All available leaf buds on this plant were marked and inspected regularly. Batches of 18, 14, 19, 24, and 15 fresh leaves on various twigs were selected that had bud breaks on April 25, April 28, May 2, May 11, and May 15, respectively. Three days after their individual bud break, leaves were treated once with gum arabic (see below) to remove the epicuticular waxes from the entire adaxial surface, thus defining d 0 in the time course. After a defined number of days the same leaf surfaces were treated a second time with gum arabic to sample epicuticular waxes for quantitative and qualitative chemical analyses. Individual leaves were sampled and analyzed separately. At each sampling time the epicuticular wax film on the entire adaxial surface was probed for six individual leaves in parallel. Wax development and leaf growth were monitored for 85 d after bud break. Data are shown for the first 57 d of development.

Surface areas of all the investigated leaves had to be monitored individually at respective sampling days without disturbing the leaf or touching the adaxial surface. Because the overall shape of leaves did not change markedly during development, digital representations of model leaf outlines could be interpolated by superposition of multiple leaves of varying age. Only the length and the width of the leaves used for wax sampling then were measured and used to reconstruct computer images of the leaves and calculate their surface areas. Based on these calculated leaf areas, wax coverages per leaf were finally determined for all sampling times. To corroborate the calculation results, surface areas of all investigated leaves were additionally determined gravimetrically after the last wax sampling using photocopies of the leaves. The surface areas of all individual leaves determined with this method were 17% ± 4% larger than the calculated values used in the present investigation.

Small pieces (0.1-0.5 cm²) of the gum arabic films removed from the adaxial surfaces of leaves at various developmental stages (see below) were used to assess epidermal cell densities. The partially translucent polymer films were viewed at 20-fold magnification under a light microscope (Zeiss Axioplan, Oberkochen, Germany). Cuticular ridges along the anticlinal walls of epidermal cells gave dark contrasts indicating the cell outlines. These contours were used to count epidermal cells within predefined areas (0.2 × 0.2 mm) and calculate cell densities. In parallel, Collodion replicas of adaxial surfaces of other leaves were prepared at various developmental stages. Their investigation yielded cell densities identical to those determined with gum arabic. With both methods only the central part of the leaf surface was probed to minimize fluctuation that could be caused by differential termination of cell division and expansion in various parts of the leaf.

Mechanical Wax Removal

A polymer film of gum arabic was employed for the selective preparation and analysis of epicuticular waxes. 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. Approximately 0.1 mL of a 90% (w/w) aqueous solution of pretreated gum were applied per centimeter² of leaf surface using a small paintbrush. After 1 to 2 h, a dry and stable polymer film had formed that could be broken off in pieces. These were collected and transferred into a vial containing 7 mL each of chloroform and water. The polymer films either from five 3.1-cm² discs (cut from mature leaves) or from entire adaxial surfaces of intact individual leaves were pooled into the same two-phase system. Tetracosane was added as internal standard, after vigorous agitation and phase separation the organic solution was removed and the solvent was evaporated under reduced pressure.

Alternatively, either frozen glycerol or water was employed for the selective removal and analysis of epicuticular waxes as described elsewhere (Jetter et al., 2000). After treatment with either glycerol or gum arabic, the leaf discs and leaves were still physically intact and could be used in repeated mechanical removal experiments or for superficial solvent extraction of the remaining cuticular waxes.

Wax Extraction

Selective superficial extraction of cuticular waxes from the adaxial surface was achieved by placing the intact leaf onto a flexible rubber mat, gently pressing a glass cylinder with 10-mm diameter onto the exposed surface, and filling the cylinder with approximately 1.5 mL of chloroform. The solvent was agitated for 30 s by pumping with a Pasteur pipette and removed. This procedure was repeated once and both extracts were combined. When any solvent leaked between cylinder and leaf surface the sample was discarded. Extracts from five individual leaves were pooled for further analysis. Tetracosane was immediately added to all the extracts of cuticular waxes as internal standard and the solvent was removed under reduced pressure.

Chemical Analysis

Prior to GC analysis, hydroxyl-containing compounds in all samples were transformed to the corresponding trimethylsilyl derivatives by reaction with bis-N,O-trimethylsilyltrifluoroacetamide (Macherey-Nagel, Düren, Germany) in pyridine (30 min at 70°C). The composition of the mixtures was studied by capillary GC (5890 II, Hewlett-Packard, Avondale, PA) with on-column injection (Hewlett-Packard 30-m OV-1 WCOT i.d. 320 µm, Chrompack, Middelburg, The Netherlands) and mass spectrometric detector (70 eV, m/z 50-650, and Hewlett-Packard 5971). GC was carried out with temperature programmed injection at 50°C, oven 2 min at 50°C, 40°C min¹ to 200°C, 2 min at 200°C, 3°C min¹ to 320°C, 30 min at 320°C, and He carrier gas inlet pressures programmed 5 min at 50 kPa, 3 kPa min¹ to 150 kPa, and 30 min at 150 kPa. Wax components were identified by comparison of their mass spectra with those of authentic standards and literature data. For quantification of individual compounds, GC was used under conditions as described above, but with carrier gas H₂ (5 min at 5 kPa, 3 kPa min¹ to 50 kPa, and 30 min at 50 kPa) and flame-ionization detector.

SEM

Epicuticular wax films were removed from the adaxial surface of P. laurocerasus leaves employing either frozen water as a cryo-adhesive (Jetter et al., 2000) or gum arabic as described above. Cryo-adhesive preparations were placed bottom-down on glass slides and air dried. This procedure exposed the original outer surface for inspection by SEM. Gum arabic treatments alternatively were placed bottom-up on glass slides, thus allowing to view the original inner surface of the epicuticular wax film. The treated leaves and the glass slides carrying the wax preparations were mounted on aluminum holders, sputtered with 20 nm of gold (Balzers Union Sputtering Device, 25 mA, 300 s), and investigated by SEM (Zeiss DSM 962, 15 kV, 6 mm).