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.

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.

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.

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.

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.

Systematics study through scanning electron microscopy; a tool for the authentication of herbal drug Mentha suaveolens Ehrh.

In all over the world, herbal drugs are usually adulterated with similar species or varieties due to incorrect identification. Most of herbal products devoid purity and quality, therefore an attempt was carried out to identify plant species and authenticate its herbal drug products from Mentha suaveolens. Microscopy tools provide an excellent platform to identify plants at species level. In this study, microscopic and pharmacokinetic parameters of M. suaveolens were observed. Plant species were collected from high diverse areas of Northern Pakistan. Macro and micro-morphology including palynology and anatomical features were analyzed to study M. suaveolens. Species characteristics were studied, while implementing microscopic techniques for the delimitation and identification of the species. Traditionally Mentha species are used to cure several diseases that is, digestive disorders, respiratory disorders. Micromorphology (stem, leaves, flowers structure, length etc.), palynology (shape, size of pollen etc.), and anatomical characters (types of stomata, epidermal cell shape, and trichomes) were studied. Micromorphology and anatomical characters were of great interest and significance to discuss the taxonomy of the species. Taxonomic characters were studied to characterize and authenticate the species. The aim of the present study is to observe in detail the taxonomic identification of the species in term of morphology, palynology, and foliar epidermal anatomy for the correct identification along with their medicinal uses in the area.

Active rearrangements in the cell wall follow polymer concentration during postharvest withering in the berry skin of Vitis vinifera cv. Corvina.

During grape postharvest withering, a worldwide practice used to produce important high-quality wines, the solute concentration increases due to dehydration, and many organoleptic and quality traits, especially related to the berry skin, are affected in a cultivar-specific manner. Nevertheless, a complete comprehension of the underlying processes is still lacking. In this work, we applied ATR-FTIR micro-spectroscopy combined with PCA to monitor cell wall biochemical changes at three stages during postharvest withering on the internal and external sides of the berry skin of the Vitis vinifera cv. Corvina, an important local variety of the Verona province in Italy. The obtained results were integrated by profiling xylogucans and pectins through immunohistochemistry and by genome-wide transcriptomic analysis performed at the same withering stages. Our analysis indicates a gradual passive polymer concentration due to water loss in the first two months of postharvest withering, followed by active structural modifications in the last month of the process. Such rearrangements involve xyloglucans in the internal surface, cuticle components and cellulose in the external surface, and pectins in both surfaces. Moreover, by investigating the expression trend of cell wall metabolism-related genes, we identified several putative molecular markers associated to the polymer dynamics. The present study represents an important step towards an exhaustive comprehension of the postharvest withering process, which is of great interest from both the biological and technological points of view.

Microscopic investigations of some selected species of Papilionaceae through SEM and LM from Skardu valley, northern Pakistan.

Pollen morphology of 10 species and foliar epidermal anatomy of eight species of Papilionaceae from Skardu valley, northern Pakistan has been estimated for the first time. The present study was commenced with an aim to provide a detailed account of the pollen morphology by scanning electron microscopy and foliar epidermal anatomy by light microscopy. The pollen aperture was tricolporate with reticulate exine in the selected species. Stomata types are actinocytic, paracytic, and anomocytic. Irregular or polygonal with undulate or straight walls, epidermal cells were reported. A unique diversity was observed in the foliar trichomes that show the taxonomic significance of the discrimination of taxa. Non-glandular trichomes were observed in the selected species which are unicellular with thin, long and pointed apical cells. Pollen and foliar micro morphological characters proved to be helpful for the identification of taxa at a specific level.

Using multiple microscopic techniques for the comparative systematic of Spergula fallax and Spergula arvensis (Caryophyllaceae).

In this study, comparative morphology, foliar anatomy and palynology of Spergula fallax and Spergula arvensis (Caryophyllaceae) were studied using multiple microscopic techniques. Genus Spergula includes worldwide five species, while in Flora of Pakistan the genus has two species. In this research, the comparative morphological, anatomical, and palynological characters of the two Pakistani Spergula species were studied. We examined some distinguishing morphological features, in both species, such as plant size, habitat, leaf morphological characters, inflorescences, flowers outer whorls, sepals and petals, and flowers number. These characters species were studied analyzing their comparative systematic significant. The foliar anatomical features also provided distinctive characters as the epidermal cell shape, the wall of the epidermal cell, lobes per cell. The differences in quantitative characters were also examined. The palynological characters showed difference in echini arrangement, echini density, and numbers of pore. Quantitative characters were variations in size of polar, equatorial, exine thickness, pore length, and width and P/E ratio. The multiple microscopic techniques provided sufficient evidence about the systematics of the genus Spergula. Based on morphological, anatomical, and palynological characters, analytical keys were developed for the identification and distinction of the species S. fallax and S. arvensis.

Systematic approach to the correct identification of Asplenium dalhousiae (Aspleniaceae) with their medicinal uses.

In present study, multiple microscope techniques were used for the systematics identification of the species Asplenium dalhousiae. The plant was collected from different phytogeographical and its natural habitat of Pakistan, where it shows higher diversity. Morphology, foliar epidermal anatomy, and spore morphological characters of the species were studied in detailed using multiple microscopic techniques through light microscopy (LM) and scanning electron microscopy (SEM). LM and SEM were used for the systematics identification of the species. Traditionally, the species is used in the ailment of many diseases, so the spore morphology, anatomical features, and morphological characters are relevant to describe the species taxonomy. The importance of multiple methods of taxonomic study (e.g., documentation and morphological characteristics) for characterizing herbs are important step in systematic certification to maintain the efficacy of herbal medicines. The aim of the present study is to examine the morphological, anatomical, and spore morphology of the species A. dalhousiae in more detailed for the correct taxonomic identification and their medicinal validation from Pakistan.

Palyno-anatomical studies of monocot taxa and its taxonomic implications using light and scanning electron microscopy.

Palyno-anatomical study of monocots taxa using Light and Scanning Electron Microscopy (SEM) was first time conducted with a view to evaluating their taxonomic significance. Studied plants were collected from different eco-climatic zones of Pakistan ranges from tropical, sub-tropical, and moist habitats. The aim of this study is to use palyno-anatomical features for the correct identification, systematic comparison, and investigation to elucidate the taxonomic significance of these features, which are useful to taxonomists for identifying monocot taxa. A signification variation was observed in quantitative and qualitative characters by using the standard protocol of light microscopy (LM) and SEM. Epidermal cell length varied from maximum in Allium griffthianum (480 ± 35.9) µm at the adaxial surface to minimum in Canna indica (33.6 ± 8.53) µm on abaxial surface. Maximum exine thickness was observed in Canna indica (4.46) µm and minimum in Allium grifthianum (0.8) µm. Variation was observed in shape and exine ornamentation of the pollen, shape of the epidermal cell, number, size, and type of stomata, guard cell shape, and anticlinal wall pattern. Based on these palyno-anatomical features a taxonomic key was developed, which help in the discrimination of studied taxa. In conclusion, LM and SEM pollen and epidermal morphology is explanatory, significant, and can be of special interest for the plant taxonomist in the correct identification of monocots taxa.

The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua.

The glandular secretory trichomes (GSTs) on Artemisia annua leaves have the capacity to secrete and store artemisinin, a compound which is the most effective treatment for uncomplicated malaria. An effective strategy to improve artemisinin content is therefore to increase the density of GSTs in A. annua. However, the formation mechanism of GSTs remains poorly understood. To explore the mechanisms of GST initiation in A. annua, we screened myeloblastosis (MYB) transcription factor genes from a GST transcriptome database and identified a MIXTA transcription factor, AaMIXTA1, which is expressed predominantly in the basal cells of GST in A. annua. Overexpression and repression of AaMIXTA1 resulted in an increase and decrease, respectively, in the number of GSTs as well as the artemisinin content in transgenic plants. Transcriptome analysis and cuticular lipid profiling showed that AaMIXTA1 is likely to be responsible for activating cuticle biosynthesis. In addition, dual-luciferase reporter assays further demonstrated that AaMIXTA1 could directly activate the expression of genes related to cuticle biosynthesis. Taken together, AaMIXTA1 regulated cuticle biosynthesis and prompted GST initiation without any abnormal impact on the morphological structure of the GSTs and so provides a new way to improve artemisinin content in this important medicinal plant.

Connecting the Molecular Structure of Cutin to Ultrastructure and Physical Properties of the Cuticle in Petals of Arabidopsis.

The plant cuticle is laid down at the cell wall surface of epidermal cells in a wide variety of structures, but the functional significance of this architectural diversity is not yet understood. Here, the structure-function relationship of the petal cuticle of Arabidopsis (Arabidopsis thaliana) was investigated. Applying Fourier transform infrared microspectroscopy, the cutin mutants long-chain acyl-coenzyme A synthetase2 (lacs2), permeable cuticle1 (pec1), cyp77a6, glycerol-3-phosphate acyltransferase6 (gpat6), and defective in cuticular ridges (dcr) were grouped in three separate classes based on quantitative differences in the ν(C=O) and ν(C-H) band vibrations. These were associated mainly with the quantity of 10,16-dihydroxy hexadecanoic acid, a monomer of the cuticle polyester, cutin. These spectral features were linked to three different types of cuticle organization: a normal cuticle with nanoridges (lacs2 and pec1 mutants); a broad translucent cuticle (cyp77a6 and dcr mutants); and an electron-opaque multilayered cuticle (gpat6 mutant). The latter two types did not have typical nanoridges. Transmission electron microscopy revealed considerable variations in cuticle thickness in the dcr mutant. Different double mutant combinations showed that a low amount of C16 monomers in cutin leads to the appearance of an electron-translucent layer adjacent to the cuticle proper, which is independent of DCR action. We concluded that DCR is not only essential for incorporating 10,16-dihydroxy C16:0 into cutin but also plays a crucial role in the organization of the cuticle, independent of cutin composition. Further characterization of the mutant petals suggested that nanoridge formation and conical cell shape may contribute to the reduction of physical adhesion forces between petals and other floral organs during floral development.

DEFECTIVE KERNEL1 (DEK1) Regulates Cell Walls in the Leaf Epidermis.

The plant epidermis is crucial to survival, regulating interactions with the environment and controlling plant growth. The phytocalpain DEFECTIVE KERNEL1 (DEK1) is a master regulator of epidermal differentiation and maintenance, acting upstream of epidermis-specific transcription factors, and is required for correct cell adhesion. It is currently unclear how changes in DEK1 lead to cellular defects in the epidermis and the pathways through which DEK1 acts. We have combined growth kinematic studies, cell wall analysis, and transcriptional analysis of genes downstream of DEK1 to determine the cause of phenotypic changes observed in DEK1-modulated lines of Arabidopsis (Arabidopsis thaliana). We reveal a novel role for DEK1 in the regulation of leaf epidermal cell wall structure. Lines with altered DEK1 activity have epidermis-specific changes in the thickness and polysaccharide composition of cell walls that likely underlie the loss of adhesion between epidermal cells in plants with reduced levels of DEK1 and changes in leaf shape and size in plants constitutively overexpressing the active CALPAIN domain of DEK1. Calpain-overexpressing plants also have increased levels of cellulose and pectins in epidermal cell walls, and this is correlated with the expression of several cell wall-related genes, linking transcriptional regulation downstream of DEK1 with cellular effects. These findings significantly advance our understanding of the role of the epidermal cell walls in growth regulation and establish a new role for DEK1 in pathways regulating epidermal cell wall deposition and remodeling.

Ultrastructure of the Epidermal Cell Wall and Cuticle of Tomato Fruit (Solanum lycopersicum L.) during Development.

The epidermis plays a pivotal role in plant development and interaction with the environment. However, it is still poorly understood, especially its outer epidermal wall: a singular wall covered by a cuticle. Changes in the cuticle and cell wall structures are important to fully understand their functions. In this work, an ultrastructure and immunocytochemical approach was taken to identify changes in the cuticle and the main components of the epidermal cell wall during tomato fruit development. A thin and uniform procuticle was already present before fruit set. During cell division, the inner side of the procuticle showed a globular structure with vesicle-like particles in the cell wall close to the cuticle. Transition between cell division and elongation was accompanied by a dramatic increase in cuticle thickness, which represented more than half of the outer epidermal wall, and the lamellate arrangement of the non-cutinized cell wall. Changes in this non-cutinized outer wall during development showed specific features not shared with other cell walls. The coordinated nature of the changes observed in the cuticle and the epidermal cell wall indicate a deep interaction between these two supramolecular structures. Hence, the cuticle should be interpreted within the context of the outer epidermal wall.

Foliar micro-morphology of Gasteria bicolor haw. (Asphodelaceae) from South Africa.

BACKGROUND: The succulent genus, Gasteria, which comprises 16 species, is endemic to South Africa and has its main centre of distribution in the Savanna Region of the Eastern Cape. Whereas G. bicolor has been investigated phyto-chemically and pharmacologically, not much data concerning the anatomical and micro-morphological features can be found in literature. MATERIALS AND METHODS: This study was undertaken, using light and scanning electron microscopy to obtain information on the micro-morphological features of this important medicinal plant to facilitate its identification and authentication. The elemental composition of the leaf was determined by energy dispersive X-ray spectroscopy (EDXS). RESULTS: The epidermal cells are either hexagonal or pentagonal in form, and are compactly arranged with undulate anti-clinal cell walls. The epidermal cell width was approximately 50 µm. Stomata apertures are elliptical and the upper epidermis of the leaf has paracytic stomata which are slightly raised above the epidermal surface with 4 to 5 subsidiary cells surrounding each stoma. Based on the EDXS microanalysis, the mineral crystals present at the level of the mesophyll of G. bicolor were probably mixtures of calcium oxalate, calcium sulphate and silica. CONCLUSION: The co-occurrence of aluminum suggests the potential role of the crystals in detoxification of aluminum and heavy metals, as reported previously.

Metal uptake and distribution in cultured seedlings of Nerium oleander L. (Apocynaceae) from the Río Tinto (Huelva, Spain).

Nerium oleander L. (Apocynaceae) is a micro-nano phanerophyte that grows in the riverbanks of the Río Tinto basin (Southwest Iberian Peninsula). The waters and soils of the Río Tinto area are highly acidic and have high concentrations of heavy metals. In this environment, N. oleander naturally grows in both extreme acidic (EA) and less extreme acidic (LEA) water courses, excluding, and bioindicating certain metals. In this work, we compared and evaluated the accumulation preferences and capacities, the distribution and processes of biomineralization of metals (Fe, Cu, Zn, Mn, Mg, Ca) in the first stages of growth of EA and LEA oleanders by means of inductively coupled plasma-mass spectrometry, scanning electron microscopy, and energy dispersive X-ray analyzer analysis. Seeds from both environments were grown and treated with a self-made solution simulating the most extreme red waters from the Río Tinto. LEA plants drastically reduces the metal uptake at the beginning, but later reactivates the uptake reaching concentration values in the same range as the EA plants. The results showed high Mn, Zn and Mg concentrations, accumulation of Fe and Cu in plants from both environments, differing from the metal concentrations of field-grown oleanders. Iron bioformations with traces of other metals were present inside and over epidermal cells and inside vascular cells of stems and roots. They were absent of leaves. The accumulation properties of N. oleander in its early stages of development make it a species to take in consideration in phytoremediation processes but optimized conditions are needed to ensure enough biomass production.

Characterizing microscale biological samples under tensile loading: stress-strain behavior of cell wall fragment of onion outer epidermis.

PREMISE OF THE STUDY: The results of published studies investigating the tissue-scale mechanical properties of plant cell walls are confounded by the unknown contributions of the middle lamella and the shape and size of each cell. However, due to their microscale size, cell walls have not yet been characterized at the wall fragment level under tensile loading. It is imperative to understand the stress-strain behavior of cell wall fragments to relate the wall's mechanical properties to its architecture. • METHODS: This study reports a novel method used to characterize wall fragments under tensile loading. Cell wall fragments from onion outer epidermal peels were cut to the desired size (15 × 5 µm) using the focused ion beam milling technique, and these fragments were manipulated onto a microelectromechanical system (MEMS) tensile testing device. The stress-strain behavior of the wall fragments both in the major and minor growth directions were characterized in vacuo. • KEY RESULTS: The measured mean modulus, fracture strength, and fracture strain in the major growth direction were 3.7 ± 0.8 GPa, 95.5 ± 24.1 MPa, and 3.0 ± 0.5%, respectively. The corresponding properties along the minor growth direction were 4.9 ± 1.2 GPa, 159 ± 48.4 MPa, and 3.8 ± 0.5%, respectively. • CONCLUSIONS: The fracture strength and fracture strain were significantly different along the major and minor growth directions, the wall fragment level modulus of elasticity anisotropy for a dehydrated cell wall was 1.23, suggesting a limited anisotropy of the cell wall itself compared with tissue-scale results.

Multiple metal accumulation within a manganese-specific genus.

PREMISE OF THE STUDY: Plants that strongly accumulate metals may be practically beneficial, and also serve as novel resources for increasing fundamental understanding of plant biology. Australian Gossia (Myrtaceae) species are delineated by a conspicuous affinity for the heavy metal manganese (Mn), which is a micronutrient crucial to photosynthesis. This genus includes several Mn hyperaccumulators such as G. bidwillii. Unusually, in G. bidwillii foliar Mn is most highly concentrated in photosynthetic cells, an observation thus far restricted to foliar-Mn accumulation in Mn hyperaccumulators. Recent discovery that several of these Gossia species accumulate other metals in addition to Mn will enable investigation as to whether primary sequestration of metals in photosynthetic tissues is restricted to Mn. METHODS: Gossia species known to accumulate nickel (Ni) or aluminum (Al) in addition to Mn were sampled in the field. Complementary proton- and electron-probe data were combined to evaluate in vivo microdistribution patterns of excessively accumulated foliar metals. KEY RESULTS: It was discovered that in addition to Mn and Ni, Gossia fragrantissima accumulated foliar zinc (Zn) and cobalt (Co), with Mn, Ni, and Co most highly localized in mesophyll cells and Zn primarily located in the upper epidermis. In G. hillii, Mn and Al were highly concentrated in the palisade and epidermis, respectively. CONCLUSIONS: This investigation provides evidence that the primary disposal of excess foliar metals in photosynthetic cells is not exclusive to Mn. It offers rare intrageneric perspective on metal compartmentation, pointing to significant variation among tonoplastal metal transporters associated with detoxification.

Terpenoid biosynthesis in trichomes--current status and future opportunities.

Glandular trichomes are anatomical structures specialized for the synthesis of secreted natural products. In this review we focus on the description of glands that accumulate terpenoid essential oils and oleoresins. We also provide an in-depth account of the current knowledge about the biosynthesis of terpenoids and secretion mechanisms in the highly specialized secretory cells of glandular trichomes, and highlight the implications for metabolic engineering efforts.