Epicuticular Wax Structure Research Articles

Sequences of epicuticular wax structures along stems in four selected tree species

Abstract Wax layer formation accompanies the processes of epidermis and cuticle formation. To examine these changes, observationsalong current-year long shoots of four woody species (Acer negundo, A. rufinerve, Gymnocladus dioica, and Gingko biloba) were made. Long shoots are suitable objects for such observations, because from the same stem, several samples can be obtained that represent a well-defined sequence of fragments of different ages. The data that were obtained from different and sometimes distant stem regions suggest that epicuticular wax crystals appear on the stems very early and quickly, since they are found near the stem apex during intensive plant growth. This was confirmed for the four examined species. Some differences, however, were noted. Very rapid production of prominent wax crystals was observed in stems. In an experiment involving mechanical wax removal from the A. negundo stem surface and subsequent analysis of the surface after several days via scanning electron microscopy (SEM), it was shown that wax regenerates very quickly. After only several hours, new emerging crystals were observed and their morphologies were essentially the same. Nearly full regeneration was observed several days after removal.

LEAF MORPHOLOGY AND ANATOMY IN THE GENUS FRAGARIA: IMPLICATIONS FOR RESISTANCES

For the first time, the genus of Fragaria was investigated by electron microscopy with regard to epicuticular wax structures and trichome formation on adaxial and abaxial leaf surfaces. In addition, cross-sections and volatile patterns of leaves of several genotypes were analyzed. Distinct differences of epicuticular wax structures among the species were observed on the adaxial but not on the abaxial leaf side. Obviously, epicuticular wax structures are not essential for susceptibility reactions to fungal diseases. Epidermal wall thickness as a reason for resistance to leaf diseases can only be supported for individual examples. Resistance to spider mite (Tetranychus urticae Koch) can be traced back to trichome formation on the abaxial side of the leaf. Additionally, the volatile analysis revealed the release of VOCs as a response to the spider mite attack.

Changes of epicuticular wax induced by enhanced UV-B radiation impact on gas exchange in Brassica napus

The epicuticular wax covering on plant surface plays important roles in protecting plants against UV radiation. However, the role of epicuticular wax in affecting leaf gas exchange under enhanced ultraviolet-B (UV-B) radiation remains obscure. In the present study, different aged leaves of Brassica napus were used to analyze the responses of crystal structure and chemical constituents of epicuticular wax to UV-B radiation and the effects of such responses on gas exchange indices. Enhanced UV-B radiation significantly decreased the amount of esters in all leaves except the first leaf, amount of secondary alcohols in the second, third and fourth leaves, and amount of primary alcohols in the second and third leaves, while increased the amounts of ketones and aldehydes in the first leaf. Enhanced UV-B level had no significant effect on the amounts of alkanes and total wax in all leaves. Exposure to UV-B radiation resulted in wax fusion on adaxial leaf and stomata opening on abaxial leaf. Fusions of plates and rods on adaxial leaf surface covered most of the stomata, thereby influencing the photosynthesis in the upper mesophyll of leaves. Enhanced UV-B level significantly reduced the net photosynthesis rate (PN) but increased the stomata conductance (gs), concentrations of intercellular CO2 (Ci), and transpiration rate (E) in all leaves. Both UV-B radiation and the wax fusion induced by enhanced UV-B radiation resulted in different stomata status on abaxial and adaxial leaf surface, causing decrease of PN, and increase of gs, Ci and E in leaves.

Epicuticular wax structures on stems and comparison between stems and leaves – A survey

The cuticle, forming the outermost layer of plant tissues and being in direct contact with the environment, consists of waxes and cutin. Waxes are hydrophobic substances that are divided in two groups: intra- and epicuticular, depending on their localisation. Epicuticular waxes appear as smooth coverings, however, many plants also produce superimposed wax structures of a crystalline nature. While studies of waxes have almost exclusively focused on leaves, here a survey of epicuticular wax structures on stems is presented. The stem surface of 343 higher plant taxa, representing 80 families, was examined using scanning electron microscopy. The adaxial and abaxial surfaces of leaves of 319 taxa were also examined to determine the relationship between wax structures on stems and leaves. Wax structures are classified, described and discussed. The results of the study indicate that stems exhibit the same main wax crystal types that have been described for leaves. Seventy percent of the examined taxa produced wax crystals on their stems. In ∼24% of the taxa, wax crystals were absent on leaves and found only on stems. In plant taxa that produce wax crystals, 40% exhibit the same type on either side of their leaves and on their stem. However, a much stronger morphological similarity exists between crystal shapes present on the adaxial and abaxial surfaces of leaves than between those present on the stem and those on leaves. In general, these observations suggest that stems are quite different than leaves in terms of their epicuticular wax structures.

Micromorphology of cactus-pear (Opuntia ficus-indica (L.) Mill) cladodes based on scanning microscopies

Cladode ultrastructural features of two prickly and two spineless Opuntia ficus-indica cultivars were examined using environmental scanning electron and atomic force microscopies. Observations focused on cladode as well as spine and glochid surface micromorphologies. Prickly cultivars were characterized by abundant cracked epicuticular wax deposits covering the cladode surface, with an amorphous structure as observed by AFM, while less abundant waxy plates were observed by ESEM on spineless cultivar cladodes. Further AFM observations allowed a rough granular and crystalloid epicuticular wax structure to be distinguished in spineless cultivars. Regarding spine micromorphology, prickly cultivars had strong persistent spines, observed by ESEM as a compact arrangement of oblong epidermal cells with a rough granular structure. However, deciduous spines in spineless cultivars had a broken transversely fissured epidermis covering a parallel arrangement of fibres. Through AFM, the deciduous spine surface presented an irregular hilly and smooth microrelief while persistent spines exhibited rough helical filamentous prints.ESEM and AFM studies of cladode surfaces from prickly and spineless cactus pear cultivars revealed valuable micro-morphological details that ought to be extended to a large number of O. ficus-indica cultivars.

Leaf surface characterization of the Tabu-No-Ki tree Machilus thunbergii using electron microscopy and white light scanning interferometry

Leaf surface features were investigated in the Tabu-No-Ki tree Machilus thunbergii by electron microscopy and white light scanning interferometry. Mature leaves of the tree were air-dried at room temperature and sputter-coated with platinum. The most prominent epicuticular wax structures of M. thunbergii leaves included granules, angular rodlets and transversely ridged rodlets. Wax granules were often observed on the adaxial leaf surface. Meanwhile, the abaxial leaf surface was characterized by transversely ridged rodlets in which the ridges were perpendicular to their longitudinal axis. Having several facets, the angular rodlets were straight and grew among clusters of transversely ridged rodlets. The adaxial leaf surface of M. thunbergii appeared to be smooth and less undulating than the abaxial leaf surface. Clusters of epicuticular waxes were predominant on the abaxial leaf surface. The average heights of the adaxial and abaxial leaf surfaces were 1.79±0.58 and 3.65±0.93μm, respectively. The average roughness and root-mean-square roughness values were lower those on the adaxial surface than on the abaxial surface. These results suggest that the transversely ridged rodlets significantly contribute to the increased surface roughness on the abaxial leaf surface.

Helium Ion Microscopy of Uncoated Pine Leaves

A recently introduced helium ion microscopy (HIM) was employed to observe uncoated pine leaf specimens. Adult leaves were collected from the seedlings of Pinus densiflora and P. rigida, air-dried at room temperature, and observed by HIM without metal coating. Ovoid or round stomata and distinct Florin rings could be discerned. The epicuticular waxes were present in the epistomatal chambers and Florin rings of stomata on the leaf surface. The epicuticular waxes were mostly straight, cylindrical, and ca. 1 <TEX>${\mu}m$</TEX> in length. The epistomatal chambers of P. rigida were filled with the epicuticular waxes, whereas those of P. densiflora were not filled with the epicuticular waxes. Based on their micromorphology, the epicuticular wax structures of the pine species were identified as tubules. These results suggest that the HIM could be used for the investigation of the plant stomata and epicuticular waxes of uncoated plant leaves. Due to the smaller ion probe and interaction volume, the HIM has advantages over conventional field emission scanning electron microscopy in terms of image resolution and charge neutralization.

Epicuticular Waxes and Stomata of Adult Scale Leaves of the Chinese Juniper Juniperus chinensis

Leaf surface structures were investigated in the Chinese juniper Juniperus chinensis by scanning electron microscopy. Adult scale leaves were collected from the tree, air-dried at room temperature, and sputter-coated with gold without further specimen preparation. Approximately fi ve stomata were locally distributed and arranged in clusters on the leaf surface. Stomata were ovoid and ca. 40 <TEX>${\mu}m$</TEX> long. The epicuticular wax structures of J. chinensis leaves were tubules and platelets. Numerous tubules were evident on the leaf regions where stomata were found. The tubules were cylindrical, straight, and ca. 1 <TEX>${\mu}m$</TEX> in length. They almost clothed the stomatal guard cells, and occluded the slit-shaped stomatal apertures. Moreover, the wax ridges were flat crystalloids that were connected to the surface by their narrow side. They did not have distinct edges, and their width/height ratio varied. In particular, the wax ridges could be discerned on the leaf regions where stomata were not present nearby. Since the wax ridges did not have distinct edges on their margin, they were identified as platelets. Instances were noted where platelets were oriented either parallel to each other or perpendicular to the cuticle surface. These results can be used in biomimetics to design the hierarchical structures for mimicking the plant innate properties such as hydrophobicity and self-cleaning effects of the leaf surface.

Wettability of biomimetic thermally grown aluminum oxide coatings

In this paper, wettability behavior of a rough but intrinsically hydrophilic oxide ceramic, formed via simple thermal oxidation of a commercial metallic alloy in laboratory air, has been analyzed. Drop shape analysis (DSA) revealed static water contact angles for the rough ceramic surfaces up to 128° (greater than for Teflon™). We propose the high apparent contact angles to be a result of surface roughening via the morphological changes of the oxide scale with oxidation conditions. The surface morphological changes occurring during the growth of the oxide film resulted in the formation of vertical platelets that ably shifted the wetting behavior from a Wenzel to an unstable Cassie–Baxter state. The platelet morphology of the ceramic resembles the structure of epicuticular waxes on certain species of superhydrophobic leaves. Moreover, surface textures for very short oxidation times were also found to increase hydrophilicity in the scale and reduce the contact angle by imparting a Wenzel state. Various characterization techniques (XRD, XPS, and SEM) were performed in order to detect the crystallographic phases in the scales, analyze carbon content and determine the morphology of the oxide layer. Morphological features of the oxide platelets were quantified and platelet width, spacing and height were found to correlate well with the apparent contact angle trend as a function of oxidation time.

Equisetum species show uniform epicuticular wax structures but diverse composition patterns

Only few data on the epicuticular waxes (EWs) of horsetails are available. This contribution therefore focuses on the wax micromorphology and chemical composition of Equisetum species of the subgenera Equisetum and Hippochaete. Distribution patterns and structural details of EW on the shoots were studied by scanning electron microscopy. After extraction with chloroform, the chemical composition of wax isolates was analysed by gas chromatography. Epicuticular wax crystals were non-oriented platelets or membraneous platelets. They were usually located on subsidiary cells of stomata and adjacent cells. Other parts of the shoots were covered mainly with a smooth wax film or small granules only. The chemical constituents found were alkanes, esters, aldehydes, primary alcohols and free fatty acids in a range of C(20)-C(36) (in esters C(36)-C(56)). All species of the subgenus Hippochaete showed a similar pattern of fractions with high percentages of alkanes and aldehydes, whereas the subgenus Equisetum species had distinctly different wax compositions. Extracts from the internodes-surfaces without well-developed EW crystals and only few stomata-showed the lowest contents of aldehydes. The covering with EW crystals will provide unhindered gas exchange and, combined with intracuticular wax, may prevent excess water loss during winter in the evergreen shoots of the subgenus Hippochaete. The results indicate that the Equisetum wax micromorphology and biosynthesis are comparable to EW of other pteridophyte classes and mosses.

Micromorphology of Epicuticular Waxes and Epistomatal Chambers of Pine Species by Electron Microscopy and White Light Scanning Interferometry

High-resolution imaging and quantitative surface analysis of epicuticular waxes and epistomatal chambers of pine species were performed by field emission scanning electron microscopy and white light scanning interferometry. Both juvenile and adult needles were collected from the two-year-old seedlings of Pinus rigida and Pinus densiflora and subjected to surface observations. Epicuticular wax structures developed on the cuticle layer as well as in the epistomatal chambers and appeared to occlude the cavities in the two pine species. The stomata of P. densiflora were characterized by more distinctly raised rings around openings than P. rigida. The most common epicuticular wax structures of the two pine species included tubules with terminal openings and coiled rodlets. Wax platelets were deposited on epistomatal chambers. Either rodlets or tubules seemed to be longer and thicker in P. rigida than those in P. densiflora. White light scanning interferometry revealed quantitative surface profiles, demonstrating more ridged (ca. 4 μm high) stomatal apertures and nearly twofold deeper (ca. 20 μm deep) epistomatal chambers of P. densiflora than those of P. rigida. These results suggest that white light scanning interferometry can be applied to unravel the quantitative surface features of epicuticular sculptures on plant leaves.

Toward in Vivo Chemical Imaging of Epicuticular Waxes

Epicuticular waxes, which are found on the outer surface of plant cuticles, are difficult to study in vivo. To monitor the growth, development, and structural alterations of epicuticular wax layers, coherent anti-Stokes Raman scattering (CARS) might be used. CARS, as a Raman-based technique, not only provides structural insight but also chemical information by imaging the spatial distribution of Raman-active vibrations. Here, we present a comparative study using CARS and scanning electron microscopy to characterize the structure of epicuticular waxes. The ability of CARS to provide detailed structural information on the biologically important wax layer was detailed on the examples of cherry laurel (Prunus laurocerasus), hoya (Hoya carnosa), and ceriman/Swiss cheese plant (Monstera sp. aff. deliciosa). We anticipate that the work presented will open a doorway for online monitoring of formation and alterations of epicuticular wax layers.

Improvement of Apple Quality and Storability by a Combination of Heat Treatment and Controlled Atmosphere Storage

The effects of two nonchemical methods [controlled atmosphere (CA) storage and postharvest heating, alone or combined] on the quality (firmness, taste, color, and skin wax) and storability (losses resulting from bruising and fungal decay) of apples were investigated in a 3-year study. Fruits of two cultivars (cv. Aroma and cv. Ingrid Marie) were mechanically wounded on two opposing sides, inoculated with conidial suspensions of one of three pathogens [Pezicula malicorticis (bull's eye rot), Penicillium expansum (blue mould), and Colletotrichum gloeosporioides (bitter rot)], exposed to 40 °C for four different exposure periods (24, 48, 72, and 96 h), and stored either in air (21.0 kPa O2 + 0.03 kPa CO2) or in CA storage (2.0 kPa O2 + 2.0 kPa CO2) for 4 months. Effect of postharvest heating on bruise susceptibility of air- or CA-stored apples was also investigated. Cultivar Aroma apples generally showed higher bruise susceptibility than cv. Ingrid Marie. The sun-exposed side of apples was less sensitive to bruising than the shaded side and red phenotypes of these two cultivars also showed increased resistance to bruising as compared with standard phenotypes. Heat treatment and CA storage, either alone or in combination, decreased bruise occurrence in both cultivars. Pz. malicorticis was the more aggressive storage pathogen for both apple cultivars followed by P. expansum and C. gloeosporioides. The highest decay severity occurred in inoculated and nonheat-treated apples stored in air. Heat treatment, especially in combination with CA storage, showed an eradicative effect on the pathogens without any negative effects on apple quality. Heat treatment maintained flesh firmness during storage, reduced ethylene production, and caused clearly visible changes in epicuticular wax structure, resulting in a higher resistance to bruising or to natural and artificial infections with the pathogens. The effective exposure period could be reduced to 24 h, because a combination of heat treatment (at 40 °C for 24 h) and CA storage showed the best protective effect against bruising and fungal decay. This combined treatment decreased bull's eye rot by 86% and 60% and bitter rot by 73% and 65% in cv. Aroma and cv. Ingrid Marie, respectively, in comparison with untreated apples.

Micromorphology of epicuticular wax structures of the garden strawberry leaves by electron microscopy: Syntopism and polymorphism

Ultrastructural aspects of leaf epicuticular wax structures were investigated in the garden strawberry Fragaria×ananassa by scanning and transmission electron microscopy. Both the adaxial and abaxial surfaces of two cultivars (Maehyang and Red Pearl) were collected and subjected to surface observations and ultrathin sections. The most prominent leaf epicuticular wax structures included membraneous platelets and angular rodlets. Most wax platelets were membraneous, and appeared to protrude from the surface at an acute angle. Angular rodlets were usually bent and had rather distinct facets in the abaxial surface of the two cultivars. Membraneous platelets were predominant on the adaxial surface of Maehyang, whereas the adaxial surface of Red Pearl was characterized by angular rodlets. However, both cultivars possessed angular rodlets on the abaxial surface, simultaneously. The combination of air-drying without vacuum and in-lens imaging of secondary electron signals with a field emission gun could impart the superb resolution at low electron dose with minimal specimen shrinkage. In vertical profiles of the leaf epidermis, epicuticular waxes were observed above the cuticle layer, and measured approximately as 50nm in thickness. The natural epicuticular waxes were seemingly mixtures of electron-dense microfibrils, and heterogeneous in shape on ultrathin sections. Distinct crystal-like strata could be hardly discernable in the wax structures. These results suggest that the garden strawberry has the nature of syntopism within one plant and polymorphism within the same species in the formation and occurrence of leaf epicuticular waxes.

Visualization of micromorphology of leaf epicuticular waxes of the rubber tree Ficus elastica by electron microscopy

Ultrastructural aspects of leaf epicuticular waxes were investigated in Ficus elastica by scanning and transmission electron microscopy. Glossy leaves of the rubber tree were collected and subjected to different regimes of specimen preparation for surface observations. F. elastica leaves were hypostomatic and stomata were surrounded with a cuticular thickening that formed a rim. The most prominent epicuticular wax structures of F. elastica leaves included granules and platelets. Without fixation and metal coating, epicuticular wax structures could be discerned on the leaf surface by low-vacuum (ca. 7 Pa) scanning electron microscopy. In terms of delineation and retention of the structures, the combination of vapor fixation by glutaraldehyde and osmium tetroxide with subsequent gold coating provided the most satisfactory results, as evidenced by high resolution and sharp protrusions of epicuticular waxes. However, erosion of epicuticular wax edges was noted in the immersion fixed leaves, showing less elongated platelets, less distinct wax edges, and granule cracking. These results suggest that the vapor fixation procedure for demonstrating three-dimensional epicuticular wax structures would facilitate characterization of diverse types of waxes. Instances were noted where epicuticular waxes grew over neighboring epidermal ridges and occluded stomata. In cross sections, epicuticular waxes were observed above the cuticle proper and ranged approximately from 100 nm to 500 nm in thickness. The native leaf epicuticular waxes had many layers of different electron density that were oriented parallel to each other and parallel or perpendicular to the cuticle surface, implying strata of crystal growth. Such retention of native epicuticular wax structures could be achieved through the use of acrylic resin treated with less harsh dehydrants and mild heat polymerization, alleviating wax extraction during specimen preparations.

The spreading and superspeading behavior of new glucosamide-based trisiloxane surfactants on hydrophobic foliage

In order to find how the molecular structure and HLB (hydrophilic–lipophilic balance) value of trisiloxane surfactant affect its wetting and spreading behavior on leaf surface, the time-dependent and the concentration-dependent spreading performance of five new glucosamide-based trisiloxane surfactants on wheat and cabbage leaf surface were investigated and compared with that of Silwet L-77 ®. The effect of different epicuticular waxes on surfactants’ spreading behavior was also investigated, and the extent of adsorption on epicuticular waxes was calculated. The results showed that the HLB values of surfactants play a very important role for showing superspreading behavior. Furthermore, the spreading performance was also affected by the molecular structure of surfactant and leaf epicuticular waxes. A new trisiloxane surfactant II show a good superspreading behavior on cabbage and wheat foliage, which is similar with that of Silwet L-77 ®. This indicated that surfactant II have a great potential to being an excellent foliage ‘superspreader’.

Impacts of greenhouse gases on epicuticular waxes of Populus tremuloides Michx.: Results from an open-air exposure and a natural O3 gradient

Epicuticular waxes of three trembling aspen (Populus tremuloides Michx.) clones differing in O3 tolerance were examined over six growing seasons (1998–2003) at three bioindicator sites in the Lake States region of the USA and at FACTS II (Aspen FACE) site in Rhinelander, WI. Differences in epicuticular wax structure were determined by scanning electron microscopy and quantified by a coefficient of occlusion. Statistically significant increases in stomatal occlusion occurred for the three O3 bioindicator sites, with the higher O3 sites having the most affected stomata for all three clones as well as for all treatments including elevated CO2, elevated O3, and elevated CO2+O3. We recorded statistically significant differences between aspen clones and between sampling period (spring, summer, fall). We found no statistically significant differences between treatments or aspen clones in stomatal frequency.

Different surface characteristics of primary and secondary needles of Pinus canariensis

Summary Surface characteristics of primary and secondary needles of Pinus canariensis were investigated using scanning electron micro-scopy and gas chromatography to study structure and composition of epicuticular wax, cuticle micromorphology and the structure of stomata. Tubular waxes could be observed on the whole needle surface of the glaucous primary needles whereas on secondary needles they were restricted to the lower surface of young needles. Recrystallization resulted in comparable wax tubes and, addition-ally, plate like structures recrystallized from primary needle wax. Isolated cuticles of primary needles were tender and showed a simple stomata complex with six subsidiary cells whereas the cuticles of secondary needles were massive and revealed 9–12 subsidiary cells. In contrast to the cuplike epistomatal chamber of the primary needles that of the secondary needles was larger and often irregularly formed. Main constituents of the cuticular wax were ω-hydroxy-n-alkanoic acids, 10-nonacosanol and n-alkanoic acids with no differences in the qualitative composition between primary and secondary needles but with some differences in the quantitative pattern. The possible role of the investigated cuticular features in adaptive strategies of the needles to avoid light and water stress is discussed.

Chinch Bug-Resistant Buffalograss: An Investigation of Tolerance, Antixenosis, and Antibiosis

Choice and no-choice studies were conducted to determine the categories (antibiosis, antixenosis, and tolerance) of resistance of four buffalograsses (NE91-118, ‘Bonnie Brae’, ‘Cody’, and ‘Tatanka’) previously identified as resistant to the western chinch bug, Blissus occiduus Barber. Antibiosis studies found no significant differences in western chinch bug fecundity, nymphal development, or survival among the resistant and susceptible buffalograsses. Tolerance studies indicated that NE91-118, Cody, and Tatanka exhibited moderate-to-high levels of tolerance based on western chinch bug damage ratings and plant height, whereas Bonnie Brae exhibited moderate-to-low levels of tolerance. Choice studies indicated the presence of antixenosis in NE91-118, whereas Cody and Tatanka showed little or no antixenosis. Scanning electron microscopy was used to disclose morphological differences between NE91-118 (resistant) and ‘378’ (susceptible). The epicuticular wax structures and trichome densities were similar between 378 and NE91-118, suggesting that morphological structures do not contribute to NE91-118 antixenosis.

Ultraviolet leaf reflectance of common urban trees and the prediction of reflectance from leaf surface characteristics

The spectral reflectance and transmittance over the wavelength range of 250–700 nm were evaluated for leaves of 20 deciduous tree species and leaf sheaths of five isogenic wax variants of Sorghum bicolor differing in visible reflectance due to cuticular waxes. Using the sorghum sheath reflectance and cuticle surface characteristics as a model, it was concluded that tree leaf reflectance above 0.06 was likely due to the presence of variously-shaped fine epicuticular wax structures on the leaf surface. Increasing the density of sub-micron wax structures corresponded to an enhanced ultraviolet (UV) reflectance over the PAR reflectance of a given leaf surface—either S. bicolor sheath or tree leaf. Amorphous globular epicuticular wax structures did not appear to scatter UV as well as wax filaments or vertical plates in varying patterns even when the dimensions of the structures were similar. Further work is needed to clarify this relationship and the influence of cellular pigments on subsurface contributions to the reflectance.