Investigations on the morpho-anatomy and histochemistry of Aconitum napellus L.

Aconitum napellus L. is a popular medicinal plant extensively used in homeopathy. This article provides detailed morphology and microscopy, including the anatomical and histochemical features of the herb, to aid authentication and quality control. In cross-section, the root in secondary growth shows the phloem surrounded by pericyclic fibers and a well-developed xylem. The stem is irregular in outline, displaying unicellular trichomes and many free collateral vascular bundles encircling the pith. The leaf is dorsiventral, hypostomatic with anomocytic and anisocytic stomata, and shows non-glandular trichomes. The floral parts are characterized by uniseriate epidermises, homogeneous mesophyll, anomocytic stomata on the abaxial surface, trichomes, and oval pollen grains. The tissue fragments in powdered herbs show these characteristics and have numerous starch grains with thimble-shaped, linear or star-shaped hilum. The detailed macroscopic and microscopic analysis provided in this study can help in the authentication and quality control of A. napellus raw materials. RESEARCH HIGHLIGHTS: Key anatomical, micromorphological, and microchemical features of Aconitum napellus are described. The results of the study can support the taxonomy of the genus Aconitum. Morphological standardization of the species reported here is helpful in the quality control of this herb.

Comparative analysis of foliar epidermal anatomical traits and their taxonomic relevance in Lilium pumilum, L. brownii, and L. davidi.

The anatomical traits of plant species are essential for taxonomic analyses and evolutionary evaluations. Clarifying the anatomical characteristics of the foliar epidermis in three distinct Lilium species L. pumilum Delile, L. brownii F.E.Br. ex Miellez and L. davidii Duch. ex Elwes were studied in this article. The objective is to assess the taxonomic relevance of these characteristics and their potential as indicators of species divergence within the genus Lilium. Plant samples were gathered in Gansu, China, from numerous populations of each species that represented a range of climatic and ecological factors. A microscopic analysis employing thin slices and peel mounts was done to assess the stomatal density and dimensions. Significant interpopulation differences in stomatal features were found in the results, offering potential opportunities for taxonomic discrimination. The species differ in qualitative and quantitative characters to differentiate the three species. The links between the observed anatomical characteristics and species classification within the Lilium genus were clarified for the three studied species. In the end, this research advances knowledge of Lilium taxonomy, aids in conservation efforts, and deepens awareness of the general patterns of plant variety. RESEARCH HIGHLIGHTS: Epidermal Traits Aid Taxonomy: Cell shape, arrangement, and structures aid Lilium Identification. Cuticle Reveals Taxonomic Clues: Thickness, composition, and structure inform classification. Micromorphology for Species ID: Cell shape, wax, and striations differentiate Lilium species.

Comparative microscopic investigations of leaf epidermis in four Ajuga species from Korea.

The genus Ajuga is widely distributed in temperate to subtropical regions, and four species are currently recognized in Korea (A. decumbens, A. multiflora, A. nipponensis, and A. spectabilis), but epidermal anatomical differences across these species have never been described. A comparative study of the leaf micromorphological characteristics of Korean Ajuga species was performed using light microscopy (LM) and scanning electron microscopy (SEM) to elucidate their taxonomic usefulness and to assess leaf micromorphological diversity. Considerable diversity in epidermal and stomatal anatomy was observed across Korean Ajuga species. Species had both hypostomatic or amphistomatic leaves, with anomocytic, anisocytic, diactyic, or actinocytic stomatal complexes. Guard cell length across species ranged from 17.66 ± 0.57 µm to 32.50 ± 2.38 µm and correlated with genome size. Abnormal stomata were frequently observed in three species (A. decumbens, A. multiflora, and A. nipponensis) but not in A. spectabilis. Three types of glandular trichomes were found: peltate in all species, short-stalked in all species, and long-stalked glandular trichomes in A. multiflora. Among the investigated leaf micromophological characters, trichome type, epidermal cell shape, and stomatal morphology were all taxonomically informative traits at a species level. RESEARCH HIGHLIGHTS: A comprehensive micromorphological description of the leaf surface is provided for Korean Ajuga species using scanning electron microscopic (SEM) and light microscopic (LM) analyses. The diverse range of stomatal development and the occurrence of polymorphic stomatal types are documented for the first time in Korean Ajuga species. The great diversity in stomatal and trichome morphology in Korean Ajuga species are taxonomically useful traits for species identification.

From macro to micro: A close-up look at Hydrangea luteovenosa and Hydrangea serrata.

Understanding the anatomical traits of the foliar epidermis is essential for making precise species identification and categorization. In this study, scanning electron microscopy (SEM) was used to examine the taxonomically significant foliar epidermal traits of Hydrangea luteovenosa and H. serrata. The qualitative and quantitative traits observed included the epidermal cell form, cuticle presence, trichome morphology, stomatal type, and guard cell features. H. serrata had a thin and smooth cuticle, and epidermal cells organized compactly into cubic or hexagonal shapes. The stomata were of the anomocytic type and dispersed, while the trichomes were straightforward, unbranched, and distributed sparsely. The guard cells had distinct cell walls and a kidney-shaped morphology. These crucial traits for taxonomy were in line with an epidermis composed of three to five layers. Similar polygonal epidermal cells with a compact arrangement were observed in H. luteovenosa, together with a thin and smooth cuticle. The stomata were anomocytic and dispersed, while the trichomes were straightforward, unbranched, and sparsely distributed. The guard cells have distinct cell walls and a kidney-shaped morphology. The traits were indicative of an epidermal structure with three to five layers. These traits helped correctly identify and categorize these two species of Hydrangea. In addition to assisting in the taxonomic classification of these species and advancing knowledge of their ecological and evolutionary links, the SEM study provided insightful information into the structural variety of these species. RESEARCH HIGHLIGHTS: Microscopic characteristics of H. luteovenosa and H. serrata Understanding the anatomical traits of the foliar epidermis is essential for precise species identification and categorization.

Taxonomic studies based on leaf epidermal microanatomy using high-resolution microscopy in Lamiaceous species and their antimicrobial effects.

The knowledge of essential oil antimicrobial activity of Lamiaceous species is assessed to describe its effects. The comprehensive foliar trichomes and stomatal morphology of the leaves of essential oil-bearing plants from the family Lamiaceae revealed diverse antimicrobial properties. The aim of this study was to investigate the foliar anatomical traits of 19 Lamiaceous taxa belonging to different tribes using light and scanning electron microscopy to correctly diagnose the species. The microanatomy of the foliar epidermis, trichomes diversity, and the stomatal apertural complex was visualized. Quantitative measurements were noted to describe the variations and the qualitative aspects for example, polygonal shape epidermal cells were examined. The stomatal aperture of four types and trichomes appendages both non-glandular and glandular was identified. Significant variation was found in both quantitative and qualitative traits, including unique ornamentation on the trichomes. The taxonomic key was constructed for accurate identification using qualitative morpho-structural traits. The outcomes of this research explored taxonomically to accurately identify the Lamiaceous species using anatomical characters. This study will provide provides the ecological adaptation linked to evolutionary traits of leaf surfaces that evolve with time to adapt the harsh environmental conditions. RESEARCH HIGHLIGHTS: Investigated foliar anatomical traits of 19 Lamiaceous species The anatomy and antimicrobial activity of essential oil yielding Lamiaceae species. SEM revealed diverse aspects including peculiar sculptured trichomes Microscopic identification of different stomatal complex.

Foliar micromorphology of selected medicinal Lamiaceae taxa and their taxonomic implication using scanning electron microscopy.

In this research, 25 medicinally used Lamiaceae species belonging to 20 genera have been studied and identified for the nine disorders. We used scanning electron microscopy (SEM) for qualitative and quantitative morphological character identification. The micromorphological characters observed here were important for distinguishing the studied taxa. The highest medicinal values were reported for Vitex negundo and Scutellaria baicalensis for all considered categories except urinary and otorhinolaryngology disorders. The foliar epidermal anatomical characteristics revealed that the micromorphological features of the Lamiaceae species provide taxonomically significant and accurate identification information to delimitate the family species. Moreover, we focused on both qualitative (epidermal cell shape, stomata type, stomatal pore shape, subsidiary cell shape, glandular trichomes, and non-glandular trichome shape) as well as quantitative features (epidermal cell size, stomata size, stomatal pore size, subsidiary cell size, and trichomes size). The trichomes diversity was different in most species' on adaxial and abaxial surfaces. In most species, anomocytic stomata were observed, but other types such as diacytic, paracytic, and tetracytic type stomata were also examined. The diverse pattern of anatomical characters suggests that the studied taxa provide insight evidence for the taxonomic observation of the Traditional Chinese Medicinal plants from the Lamiaceae. This work sets an avenue for future research and taxonomic exploration of medicinal flora through microscopic investigations. RESEARCH HIGHLIGHTS: This research offers a thorough microscopic identification of the family Lamiaceae. Taxonomic information on the trichome characters and types for the accurate authentication. Qualitative and quantitative characterization of 25 medicinally used Lamiaceae taxa.

Palyno-anatomical characters and their systematic significance in the family Apiaceae from Chitral, eastern Hindu Kush, Pakistan.

The present study was performed to provide a detailed explanation of leaf epidermal anatomy and pollen micromorphological features of selected species of family Apiaceae from Chitral, eastern Hindu Kush region as the basis of forthcoming studies. In the present article pollen morphology of eight species and foliar epidermal of seven species of family Apiaceae have been examined through microscopic techniques. In results two types of pollen prolate (five species) and perprolate (three species) with three colpi have been recorded. The exine ornamentation was found to be regulate, striate, and cerebroid. Largest pollen was found in Heracleum leucocarpum with the polar diameter of 43.25 µm and equatorial diameter of 21.6 µm. Smallest pollen was observed in Elaeosticta chitralica with the polar diameter of 18.4 µm. The P/E ratio varied from 1.59 to 2.16. Regarding to foliar epidermal anatomy, three types of epidermal cells including rectangular, irregular, and polygonal with variation in anticlinal wall pattern were determined. In the selected species three kinds of stomata comprising anisocytic, anomocytic, and paracytic type were reported in the current research. The size of epidermal cells ranged from 106 × 42.50 µm in Bupleurum falcatum subsp. cernuum and 77.25 × 26.35 µm in Prangos pabularia in adaxial surface. Largest stomatal complex was found in Prangos pabularia both in adaxial 33.55 × 20.05 µm and abaxial 50.25 × 39.40 µm. All the observed quantitative and qualitative features of the species were proved to be useful in the delimitation of species at generic and species level.

Molecular insights into the complex mechanics of plant epidermal cell walls.

Plants have evolved complex nanofibril-based cell walls to meet diverse biological and physical constraints. How strength and extensibility emerge from the nanoscale-to-mesoscale organization of growing cell walls has long been unresolved. We sought to clarify the mechanical roles of cellulose and matrix polysaccharides by developing a coarse-grained model based on polymer physics that recapitulates aspects of assembly and tensile mechanics of epidermal cell walls. Simple noncovalent binding interactions in the model generate bundled cellulose networks resembling that of primary cell walls and possessing stress-dependent elasticity, stiffening, and plasticity beyond a yield threshold. Plasticity originates from fibril-fibril sliding in aligned cellulose networks. This physical model provides quantitative insight into fundamental questions of plant mechanobiology and reveals design principles of biomaterials that combine stiffness with yielding and extensibility.

Fluctuations shape plants through proprioception.

Plants constantly experience fluctuating internal and external mechanical cues, ranging from nanoscale deformation of wall components, cell growth variability, nutating stems, and fluttering leaves to stem flexion under tree weight and wind drag. Developing plants use such fluctuations to monitor and channel their own shape and growth through a form of proprioception. Fluctuations in mechanical cues may also be actively enhanced, producing oscillating behaviors in tissues. For example, proprioception through leaf nastic movements may promote organ flattening. We propose that fluctuation-enhanced proprioception allows plant organs to sense their own shapes and behave like active materials with adaptable outputs to face variable environments, whether internal or external. Because certain shapes are more amenable to fluctuations, proprioception may also help plant shapes to reach self-organized criticality to support such adaptability.

Structure, Assembly and Function of Cuticle from Mechanical Perspective with Special Focus on Perianth.

This review is devoted to the structure, assembly and function of cuticle. The topics are discussed from the mechanical perspective and whenever the data are available a special attention is paid to the cuticle of perianth organs, i.e., sepals, petals or tepals. The cuticle covering these organs is special in both its structure and function and some of these peculiarities are related to the cuticle mechanics. In particular, strengthening of the perianth surface is often provided by a folded cuticle that functionally resembles profiled plates, while on the surface of the petal epidermis of some plants, the cuticle is the only integral continuous layer. The perianth cuticle is distinguished also by those aspects of its mechanics and development that need further studies. In particular, more investigations are needed to explain the formation and maintenance of cuticle folding, which is typical for the perianth epidermis, and also to elucidate the mechanical properties and behavior of the perianth cuticle in situ. Gaps in our knowledge are partly due to technical problems caused by very small thicknesses of the perianth cuticle but modern tools may help to overcome these obstacles.

Atomic Force Microscopy to Study Cell Wall Mechanics in Plants.

Atomic force microscopy (AFM) is an indentation technique used to reconstruct the topography of various materials and organisms. AFM can also measure the mechanical properties of the sample. In plants, AFM is applied to image cell wall structural details and measure the elastic properties in the outer cell walls. Here, I describe the use of high-resolution AFM to measure the elasticity of resin-embedded ultrathin sections of leaf epidermal cell walls. This approach allows to access the fine details within the wall matrix and eliminate the influence of the topography or the turgor on mechanical measurements. In this chapter, the sample preparation, AFM image acquisition, and processing of force curves are described. Altogether, these methods allow to measure the wall stiffness and compare different cell wall regions.

Three-dimensional reconstruction and comparison of vacuolar membranes in response to viral infection.

The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional (3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus (CMV) or tobacco necrosis virus A Chinese isolate (TNV-AC ), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1a and 2a and TNV-AC auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast. Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant (+) RNA virus replication complexes.

Comparative Analyses of the Self-Sealing Mechanisms in Leaves of Delosperma cooperi and Delosperma ecklonis (Aizoaceae).

Within the Aizoaceae, the genus Delosperma exhibits a vast diversification colonizing various ecological niches in South-Africa and showing evolutionary adaptations to dry habitats that might include rapid self-sealing. Leaves of Delosperma react to external damage by the bending or contraction of the entire leaf until wound edges are brought into contact. A study of leaf morphology and anatomy, biomechanics of entire leaves and individual tissues and self-sealing kinematics after a ring incision under low and high relative humidity (RH) was carried out comparing the closely related species Delosperma cooperi and Delosperma ecklonis, which are indigenous to semi-arid highlands and regions with an oceanic climate, respectively. For both species, the absolute contractions of the examined leaf segments ("apex", "incision", "base") were more pronounced at low RH levels. Independent of the given RH level, the absolute contractions within the incision region of D. cooperi were significantly higher than in all other segments of this species and of D. ecklonis. The more pronounced contraction of D. cooperi leaves was linked mainly to the elastic properties of the central vascular strand, which is approximately twice as flexible as that of D. ecklonis leaves.

High-Resolution Imaging of Cellulose Organization in Cell Walls by Field Emission Scanning Electron Microscopy.

Field emission scanning electron microscopy (FESEM) is a powerful tool for analyzing surface structures of biological and nonbiological samples. However, when it is used to study fine structures of nanometer-sized microfibrils of epidermal cell walls, one often encounters tremendous challenges to acquire clear and undistorted images because of two major issues: (1) Preparation of samples suitable for high resolution imaging; due to the delicateness of some plant materials, such as onion epidermal cell walls, many things can happen during sample processing, which subsequently result in damaged samples or introduce artifacts. (2) Difficulties to acquire clear images of samples which are electron-beam sensitive and prone to charging artifacts at magnifications over 100,000×. In this chapter we described detailed procedures for sample preparation and conditions for high-resolution FESEM imaging of onion epidermal cell walls. The methods can be readily adapted for other wall materials.

Scanning electron microscopic screening of 20 medicinally important Asteroideae taxa.

In the present study anatomical characterization of 20 medicinally important Asteroideae species were done under light and scanning electron microscopy. Variety of qualitative and quantitative anatomical characters like epidermal cells, stomata, guard cells, subsidiary cells, trichomes and oil droplets were observed. Generally pentagonal, polygonal, irregular or hexagonal, smooth, undulating thick walled epidermal cells were observed in studied species. In abaxial surface Thymophylla tenuiloba L. possessed the largest length of epidermal cell that is, 221.6 (156.6-286.6) µm whereas Bellis perenis L. showed the smallest length that is, 46.4 (32.6-60.2) µm. Average width of epidermal cells ranged from 57 (22-92) µm to 169 (127.9-210.1) µm. Cosmos sulphureus Cav. had smallest width while Thymophylla tenuiloba L. had the largest width. In adaxial surface Artemisia absinthium L. possessed the largest length of epidermal cell that is, 269 (165.1-372.9) µm whereas Bellis perenis L. showed the smallest length that is, 61.4 (42.6-80.2) µm. Average width of epidermal cells ranged from 50.8 (32.6-69) µm to 260 (116-202) µm. Thymophylla tenuiloba L. had smallest width while Dahlia pinnata Cav. had the largest width. Among stomatal characters anisocytic, anomocytic, and diacytic stomata were observed in selected species of Asteroideae. Nonglandular uniserate, multicellular, unbranched pointed tips with bulbous base trichomes were reported in some Asteroideae members while some possessed glandular, capitates mushroom like multicellular trichomes covered with tubercle papicles. Rounded, oval, triangular shaped oil droplets were observed in some species. It is concluded that qualitative and qualitative anatomical variations in trichomes, stomata and epidermal cells are of good taxonomic value for the Asteroideae species.

Chemical Defoliant Promotes Leaf Abscission by Altering ROS Metabolism and Photosynthetic Efficiency in Gossypium hirsutum.

Chemical defoliation is an important part of cotton mechanical harvesting, which can effectively reduce the impurity content. Thidiazuron (TDZ) is the most used chemical defoliant on cotton. To better clarify the mechanism of TDZ promoting cotton leaf abscission, a greenhouse experiment was conducted on two cotton cultivars (CRI 12 and CRI 49) by using 100 mg L-1 TDZ at the eight-true-leaf stage. Results showed that TDZ significantly promoted the formation of leaf abscission zone and leaf abscission. Although the antioxidant enzyme activities were improved, the reactive oxygen species and malondialdehyde (MDA) contents of TDZ increased significantly compared with CK (water). The photosynthesis system was destroyed as net photosynthesis (Pn), transpiration rate (Tr), and stomatal conductance (Gs) decreased dramatically by TDZ. Furthermore, comparative RNA-seq analysis of the leaves showed that all of the photosynthetic related genes were downregulated and the oxidation-reduction process participated in leaf shedding caused by TDZ. Consequently, a hypothesis involving possible cross-talk between ROS metabolism and photosynthesis jointly regulating cotton leaf abscission is proposed. Our findings not only provide important insights into leaf shedding-associated changes induced by TDZ in cotton, but also highlight the possibility that the ROS and photosynthesis may play a critical role in the organ shedding process in other crops.

Drought stress modify cuticle of tender tea leaf and mature leaf for transpiration barrier enhancement through common and distinct modes.

Cuticle is the major transpiration barrier that restricts non-stomatal water loss and is closely associated with plant drought tolerance. Although multiple efforts have been made, it remains controversial what factors shape up the cuticular transpiration barrier. Previously, we found that the cuticle from the tender tea leaf was mainly constituted by very-long-chain-fatty-acids and their derivatives while alicyclic compounds dominate the mature tea leaf cuticle. The presence of two contrasting cuticle within same branch offered a unique system to investigate this question. In this study, tea seedlings were subjected to water deprivation treatment, cuticle structures and wax compositions from the tender leaf and the mature leaf were extensively measured and compared. We found that cuticle wax coverage, thickness, and osmiophilicity were commonly increased from both leaves. New waxes species were specifically induced by drought; the composition of existing waxes was remodeled; the chain length distributions of alkanes, esters, glycols, and terpenoids were altered in complex manners. Drought treatment significantly reduced leaf water loss rates. Wax biosynthesis-related gene expression analysis revealed dynamic expression patterns dependent on leaf maturity and the severity of drought. These data suggested that drought stress-induced structural and compositional cuticular modifications improve cuticle water barrier property. In addition, we demonstrated that cuticle from the tender leaf and the mature leaf were modified through both common and distinct modes.

A Scanning Electron Micrograph-based Resource for Identification of Loci Involved in Epidermal Development in Tomato: Elucidation of a New Function for the Mixta-like Transcription Factor in Leaves.

The aerial epidermis of plants plays a major role in environmental interactions, yet the development of the cellular components of the aerial epidermis-trichomes, stomata, and pavement cells-is still not fully understood. We have performed a detailed screen of the leaf epidermis in two generations of the well-established Solanum lycopersicum cv M82 × Solanum pennellii ac. LA716 introgression line (IL) population using a combination of scanning electron microscopy (SEM) techniques. Quantification of trichome and stomatal densities in the ILs revealed four genomic regions with a consistently low trichome density. This study also found ILs with abnormal proportions of different trichome types and aberrant trichome morphologies. This work has led to the identification of new, unexplored genomic regions with roles in trichome formation in tomato. This study investigated one interval in IL2-6 in more detail and identified a new function for the transcription factor SlMixta-like in determining trichome patterning in leaves. This illustrates how these SEM images, publicly available to the research community, provide an important dataset for further studies on epidermal development in tomato and other species of the Solanaceae family.

Superrepellency of underwater hierarchical structures on Salvinia leaf.

Biomimetic superhydrophobic surfaces display many excellent underwater functionalities, which attribute to the slippery air mattress trapped in the structures on the surface. However, the air mattress is easy to collapse due to various disturbances, leading to the fully wetted Wenzel state, while the water filling the microstructures is difficult to be repelled to completely recover the air mattress even on superhydrophobic surfaces like lotus leaves. Beyond superhydrophobicity, here we find that the floating fern, Salvinia molesta, has the superrepellent capability to efficiently replace the water in the microstructures with air and robustly recover the continuous air mattress. The hierarchical structures on the leaf surface are demonstrated to be crucial to the recovery. The interconnected wedge-shaped grooves between epidermal cells are key to the spontaneous spreading of air over the entire leaf governed by a gas wicking effect to form a thin air film, which provides a base for the later growth of the air mattress in thickness synchronously along the hairy structures. Inspired by nature, biomimetic artificial Salvinia surfaces are fabricated using 3D printing technology, which successfully achieves a complete recovery of a continuous air mattress to exactly imitate the superrepellent capability of Salvinia leaves. This finding will benefit the design principles of water-repellent materials and expand their underwater applications, especially in extreme environments.

The semidominant mutation w5 impairs epicuticular wax deposition in common wheat (Triticum aestivum L.).

KEY MESSAGE: The semidominant EMS-induced mutant w5 affects epicuticular wax deposition and mapped to an approximately 194-kb region on chromosome 7DL. Epicuticular wax is responsible for the glaucous appearance of plants and protects against many biotic and abiotic stresses. In wheat (Triticum aestivum L.), ß-diketone is a major component of epicuticular wax in adult plants and contributes to the glaucousness of the aerial organs. In the present study, we identified an ethyl methanesulfonate-induced epicuticular wax-deficient mutant from the elite wheat cultivar Jimai22. Compared to wild-type Jimai22, the mutant lacked ß-diketone and failed to form the glaucous coating on all aerial organs. The mutant also had significantly increased in cuticle permeability, based on water loss and chlorophyll efflux. Genetic analysis indicated that the mutant phenotype is controlled by a single, semidominant gene on the long arm of chromosome 7D, which was not allelic to the known wax gene loci W1-W4, and was therefore designated W5. W5 was finely mapped to an ~ 194-kb region (flanked by the molecular markers SSR2 and STARP11) that harbored four annotated genes according to the reference genome of Chinese Spring (RefSeq v1.0). Collectively, these data will broaden the knowledge of the genetic basis underlying epicuticular wax deposition in wheat.