Plant surfaces have specific physicochemical properties due to the cuticle and, in particular, due to the epicuticular waxes deposited on it. In previous studies, bio-inspired technical surfaces were often used with surface properties only partially corresponding to those of the natural surfaces. In the present work, an artificial leaf surface was developed that corresponded to the plant leaf surface in its chemical properties and its wettability.

Wax morphology Natural surface

First, the surface properties of the natural wheat leaf were characterized. For wheat leaves, platelets as well as tubules have been previously described as wax structures [17,20,49-52]. Further, the wax structures can vary depending on the developmental stage of the plant [49]. SEM investigation showed platelets on all leaves of different ages (Figure 3a, Supporting Information File 1, Figure S2). This result meets the expectations, as tubules were described for other plant organs such as leaf sheets, stems, and glumes [50,51,53,54], or only on the abaxial sides of the glaucous flag leaves of plants at a later stage of development [50,51]. In our study, the structures of both leaf sides were the same (Supporting Information File 1, Figure S2). This is in accordance with other studies. In Koch et al. [20] and Wang et al. [53] plants in a comparable developmental stage were investigated. A network of densely packed wax platelets was also described for both sides of the leaf [53].

Abiotic factors such as temperature, humidity, pollution, and radiant energy might cause changes in the wax morphology [55-57]. To take this into account, wheat plants were also cultivated outdoors under natural growth conditions. No culture-specific differences in the wax morphology were observed. As with the greenhouse plants, the leaves of the plants cultivated outdoors were covered with wax platelets. Hence, the wax morphology of the investigated wheat leaves showed no developmental and ecological variability. The wax platelets could therefore be used as a native model for the development of artificial leaf surfaces.

Artificial surfaces

As a decisive prerequisite for the applicability of the artificial surface as bio-inspired leaf surface, it was defined that the artificial surface needs to have a homogeneous coating and uniform wettability. Previous recrystallization studies have shown that a homogeneous coating can be produced by thermal evaporation [40,46,58-60]. In the present work, a homogeneous coating was also achieved with this method, as can be seen in the photograph (Figure 4) and in the SEM micrograph (Figure 3). The latter showed that the wax recrystallized to granular structures with single plates between the granular network. However, after deposition of the lowest wax mass, almost no plates were formed, indicating that the polar and amorphous substrate prevents the formation of a platelet structure. In contrast, higher wax masses lead to a growth of the platelets on the wax deposited below. It was clearly visible that the individual plates were not formed directly on the substrate, but stood on a granular layer. Koch et al. [20] demonstrated that on non-polar substrates, such as highly ordered pyrolytic graphite, wax composed of primary alcohols recrystallize into platelets, as on the wheat leaves. Even though the recrystallized structures in the present study did not exactly match the native wax structures, the wetting properties of the artificial leaf surfaces were similar to those of the natural leaf. The glass substrate used here offers an easy-to-handle and inexpensive way to develop large numbers of wax-coated samples with wetting properties that are similar to that of the natural leaf.

Wax chemistry Natural surface

Next to the wax morphology, the wax chemistry of wheat leaves was analyzed, as it plays an important role in the functionality of the leaf surface. The extracted wheat wax was composed of alcohols, aldehydes, esters, and acids. The main component was 1-octacosanol (Figure 6). The results are in agreement with previous studies on the wax chemistry of other wheat varieties [20,50,61,62]. β-Diketone, which is a component of wax tubules, has so far only been detected in wax of wheat leaves at later developmental stages and other plant organs [50,53,61,63-66]. Alkanes, which were detected in wheat wax in some other studies, were not a component of the wax examined here [20,52,53,62-64].

Developmental changes can have an influence on the wax composition [67,68]. Therefore, the composition of epicuticular waxes of leaves of different ages was analyzed in the greenhouse-cultured plants to examine whether there were any differences. The total wax amount decreased from leaf 2 to leaf 4, that is, from older to younger leaves. The decrease in wax amounts was evenly distributed over all substance classes, so that there were no notable differences in the relative wax composition and the corresponding wax morphology (Supporting Information File 1, Figure S4).

Abiotic factors can have an influence on the amount of epicuticular wax. For example, previous studies have shown that higher temperatures, reduced humidity, increased UV radiation, or drought stress can lead to increased wax accumulation [55,64,69-71]. Therefore, it was also examined whether there were differences in the wax chemistry between the greenhouse plants and the outdoor plants. The results showed hardly any differences between the differently cultivated plants. The total wax amount and the amounts of the single substance classes were the same (Figure 6). Consistent with the results of the wax morphology study, the wax composition of wheat appears to be strongly genetically fixed and little influenced by environmental factors. Thus, the wax extracts of plants growing outdoors or indoors can be used for the development of artificial leaf surfaces.

Artificial surfaces

To make sure that wax composition had not changed during the coating process, the wax layer of a coated artificial leaf surface was analyzed. By comparison of the native wax with the wax recrystallized after thermal evaporation, we could show that the wax still had the same chemical composition as the wax on the native leaves. In the wax precipitate on glass, the same substances were detected in the same amounts as in the wax that was vaporized (Figure 6). Thus, no transformation of substances had taken place and the wax composition was stable. In a previous study, the wax composition of leek wax was also found to be thermally stable [72].

Since the evaporated substances spread uniformly in vacuum from the point source and the samples were placed in an arc above the substance source, in previous studies, the wax coverage was calculated approximately from the quotient of the evaporated masses and the surface area of a hemisphere [42,58,72,73]. The analysis of the wax coating of the evaporated surface performed here, in contrast, allowed for an accurate measurement of the wax coverage. At 15.7 ± 2.6 µg·cm−2, it was the same as the wax coating of a wheat leaf (Figure 6). The wax coating of the technical surface thus corresponded in its chemical properties both qualitatively and quantitatively to the wax coating of the natural surface.

Wetting properties Natural surface

The wetting behavior of a plant surface is relevant for the interaction of the plant with its biological and non-biological environment. For instance, surface moisture plays a crucial role in the development of fungal diseases in plants [74]. Also, the wettability has to be considered in the context of the application of agrochemicals [75]. Thus, as a criterion for success it can be postulated that an artificial in vitro test system should reflect the wettability of the native surface. Consequently, in addition to wax morphology and wax chemistry, the wettability of wheat leaves was investigated.

Often, only the CAs are used to describe the wettability. However, specifying CAs alone to describe wettability can be misleading and insufficient [75,76]. For example, a droplet with a high CA may stick to an inclined surface or roll off easily, depending on the wettability state. Therefore, the CAH and the TA of wheat leaves were also determined the present work.

The investigated wheat leaves were all hydrophobic (Table 1), which is in agreement with results of previous studies [29,50,77,78]. The CAH values ranged from 8.2° ± 8.3° to 25.0° ± 10.3° and the TAs ranged from 10.8° ± 7.3° to 31.3° ± 21.3° (Table 1). Wheat leaves are not vertically oriented to the plant axis. TAs provide information on droplet adhesion on such inclined surfaces. For wheat leaves, we could not find any comparative values for TAs in the literature. In the context of active ingredient application and uptake, Peirce et al. [78] investigated the retention of applied water droplets on wheat leaves. In this study, high CAs and very low CAH were also found, which indicates the ability for self-cleaning. However, the investigated plants were cultivated in the greenhouse. Weather-related damage to the leaves of the outdoor plants examined in the present study possibly caused the larger CAH and TAs and the high standard deviations.

Droplets generally rolled off rapidly from the wheat leaves. Microscopic images taken during the wetting measurements indicated that the water droplets did not completely fill the space between the anticlinal cell walls. Both observations suggest Cassie wetting or intermediate wetting. Cassie wetting was also assumed in a previous wettability study on wheat leaves [78]. In individual specimens, however, the water droplets remained attached to the surface and did not roll off even at a TA of 90°. In such samples, the wax layer was probably damaged or contaminated. Both wax alteration and contamination can lead to a change in wettability [75,79]. No damage or contamination was visible to the naked eye in the leaves used. SEM images of the leaves from the field cultures, however, showed clear damage in the wax layer (removed and flattened crystals) on the leaf surfaces in some cases (Supporting Information File 1, Figure S3). Presumably, a transition to Wenzel wetting takes place at damaged or dirty areas, so that the droplets no longer roll off. In the case of a drop on a rough surface, liquid is pressed between the cavities by the Laplace pressure. If the liquid penetrates into the cavities so far that it touches the substrate, a transition of the wetting states takes place [75]. Whether a transition occurs or not is influenced by the shape and dimensions of the supporting structures and by the droplet size [80]. In the present study, the droplet volume was always the same, but the structures determining the wetting may have been destroyed or masked by particles such that a transition could take place. To evaluate the wetting properties, it is advisable to use plants from greenhouse cultures, as their surfaces are less heterogeneous. The experiments in this work have shown that wax morphology and wax composition were barely influenced by the cultivation conditions (Figure 6). Nevertheless, plants in greenhouse cultures are better protected from phytopathogens and from wind and rain abrasion of wax. In this work, however, the wettability of outdoor grown plants was studied to be close to natural conditions.

Artificial surfaces

In this work, an artificial leaf surface was developed which, in addition to the same wax chemistry, also should have the same wetting properties as a natural leaf. This goal was achieved by vapor deposition with the medium amount of the wax extract. The surface coated in this way resembled the natural surface regarding all three studied wetting parameters (Figure 8).

The chemical analysis had shown that the wax composition of the wax coating on glass corresponds to that of the natural leaf surface. However, not only the wax chemistry but also the wax structure plays a role for the wettability. The different amounts of wax used made it possible to vary the structures and, thus, the wettability of the artificial surfaces. For example, the CA of the artificial surface covered with the small amount of evaporated wax was smaller than that of the wheat leaf. Hence, these artificial surfaces were more wettable than the natural ones (Figure 8a). Water droplets deposited did not roll off even at TAs of up to 90°. Presumably the water drops are not on the structures but sink in between them, resulting in Wenzel wetting [33]. On microstructured Si wafers, it could be shown that, depending on the height and the distance of the supporting columns of the surface, a transition from Cassie wetting to Wenzel wetting takes place. These and other criteria for the transition of a wetting stage were summarized by Bhushan and Jung [73].

The artificial surfaces vapor-deposited with 1400 and 2800 µg wax were as hydrophobic as the wheat leaves. However, the wax coating of the surface coated with 2800 µg appeared to be unstable. It seemed that rolling droplets carried wax particles on their surface and left a slight trace in the coating (data not shown). No loss of wax was observed on the artificial surface that was vapor-deposited with 1400 µg wax.

Utilization of the artificial leaf surface

Here, an artificial leaf surface was developed that mimics the physicochemical properties of a natural leaf surface. The morphology of the recrystallized structures was not a 1:1 copy of the native structures, but the properties were still transferred. This proves that the thermal evaporation process made it possible to transfer the surface properties of the native surface to the technical surface. To the best of the authors’ knowledge, no artificial surface exists to date that reflects the wetting properties in addition to the chemistry of the epicuticular waxes. In previous studies, sophisticated systems provided with wax mixtures or with individual wax components were used to investigate the germination behavior of fungal spores [62,81-83]. These had the same wax chemistry, but the wettability was higher than that of the leaf surface. Also in this study, fungal spores were applied to the artificial leaf surface as a first application test. The microscopic analysis showed that the spores germinate and differentiate on the artificial leaf surface. Even though the number of germinated spores was lower than on the natural leaf, the spores were able to germinate on our artificial leaf surface. Hence, the here developed artificial leaf surface offers an opportunity to study interactions of the plant surface ex situ. In contrast to the natural leaf, the artificial leaf surface is less complex and easily adjustable.