Layer Cell Wall Research Articles

The Nanoscale Ordering of Cellulose in a Hierarchically Structured Hybrid Material Revealed Using Scanning Electron Diffraction.

Cellulose, being a renewable and abundant biopolymer, has garnered significant attention for its unique properties and potential applications in hybrid materials. Understanding the hierarchical arrangement of cellulose nanofibers is crucial for developing cellulose-based materials with enhanced mechanical properties. In this study, the use of Scanning Electron Diffraction (SED) is presented to map the nanoscale orientation of cellulose fibers in a bio-composite material with a preserved wood cell structure. The SED data provides detailed insights into the ordering of cellulose with an extraordinary resolution of ≈15nm. It enables a quantitative analysis of the fiber orientation over regions as large as entire cells. A highly organized arrangement of cellulose fibers within the secondary cell wall is observed, with a gradient of orientations toward the outer part of the wall. The in-plane fiber rotation is quantified, revealing a uniform orientation close to the middle lamella. Transversely sectioned material exhibits similar trends, suggesting a layered cell wall structure. Based on the SED data, a 3D model depicting the complex helical alignment of fibers throughout the cell wall is constructed. This study demonstrates the unique opportunities SED provides for characterizing the nanoscale hierarchical arrangement of cellulose nanofibers, empowering further research on a range of hybrid materials.

Super-strong biomimetic bulk bamboo-based composites by a neural network interfacial design strategy

As a sustainable ecological material, bamboo has become a popular modern green building material because of its rich yield, lightweight, high strength and rich cultural heritage. However, due to the limitation of bamboo tube thickness, multiple thickness-direction laminations are usually required to achieve large-sized materials, which leads to a significant decrease in strength. Therefore, it is urgent to find a way produce high-strength bamboo engineering composites on a large scale. Herein, a neural network interface design strategy was proposed, and a mechanical dissociation and partial matrix removal pretreatment method was used to open the weak intercellular layer and bamboo cell wall layer to increase the resin permeation channels. This allowed the resin to form a multi-scale bonding interface between multiple dense bamboo layers, achieving the preparation of bulk bamboo-based composite with adjustable dimensions and properties. The neural network-like bonding interface could firmly fix the compressed bamboo cells and enhance the mechanical properties of the bamboo cell wall and intercellular layer of bamboo, resulting in a tensile strength of 853 MPa for the composite, which was nearly three times that of natural bamboo and significantly superior to many structural materials such as alloys and other bamboo-based composites. In addition, this material showed good mildew resistance, flame retardancy and dimensional stability. This large-size bamboo composites are easy to scale production, which can be used in fields such as wind turbine blades, building structures, and outdoor walkways in the future.

Functional annotation of rhizospheric phageome of the wild plant species Moringa oleifera.

The study aims to describe phageome of soil rhizosphere of M.oleifera in terms of the genes encoding CAZymes and other KEGG enzymes. Genes of the rhizospheric virome of the wild plant species Moringa oleifera were investigated for their ability to encode useful CAZymes and other KEGG (Kyoto Encyclopedia of Genes and Genomes) enzymes and to resist antibiotic resistance genes (ARGs) in the soil. Abundance of these genes was higher in the rhizospheric microbiome than in the bulk soil. Detected viral families include the plant viral family Potyviridae as well as the tailed bacteriophages of class Caudoviricetes that are mainly associated with bacterial genera Pseudomonas, Streptomyces and Mycobacterium. Viral CAZymes in this soil mainly belong to glycoside hydrolase (GH) families GH43 and GH23. Some of these CAZymes participate in a KEGG pathway with actions included debranching and degradation of hemicellulose. Other actions include biosynthesizing biopolymer of the bacterial cell wall and the layered cell wall structure of peptidoglycan. Other CAZymes promote plant physiological activities such as cell-cell recognition, embryogenesis and programmed cell death (PCD). Enzymes of other pathways help reduce the level of soil H2O2 and participate in the biosynthesis of glycine, malate, isoprenoids, as well as isoprene that protects plant from heat stress. Other enzymes act in promoting both the permeability of bacterial peroxisome membrane and carbon fixation in plants. Some enzymes participate in a balanced supply of dNTPs, successful DNA replication and mismatch repair during bacterial cell division. They also catalyze the release of signal peptides from bacterial membrane prolipoproteins. Phages with the most highly abundant antibiotic resistance genes (ARGs) transduce species of bacterial genera Pseudomonas, Streptomyces, and Mycobacterium. Abundant mechanisms of antibiotic resistance in the rhizosphere include "antibiotic efflux pump" for ARGs soxR, OleC, and MuxB, "antibiotic target alteration" for parY mutant, and "antibiotic inactivation" for arr-1. These ARGs can act synergistically to inhibit several antibiotics including tetracycline, penam, cephalosporin, rifamycins, aminocoumarin, and oleandomycin. The study highlighted the issue of horizontal transfer of ARGs to clinical isolates and human gut microbiome.

Functional groups on wheat (Triticum aestivum) root surface affect aluminium transverse accumulation

Plant root growth is inhibited markedly by aluminium (Al) even at micromolar concentration and Al is mainly accumulated in plant roots outer layer cell walls. But the underlying reason for this asymmetric transverse distribution is unknown. In this study, two wheat (Triticum aestivum L.) genotypes ET8 and ES8 differing in Al resistance were investigated by hydroculture. The Al-tolerant ET8 expressed a higher root elongation rate (RER) than Al-sensitive ES8 under Al stress. Morphological examination showed symptoms such as root surface ruptures were observed in ET8 and ES8, with ES8 being more obvious. The cation exchange capacity (CEC) values of root tips of ES8 under different Al concentrations are higher than those of ET8. The sensitive genotype ES8 accumulated more Al than ET8 in plant apical root tips as well as cell walls. Under 48 h Al exposure, the root cell wall pectin concentration was increased with a higher magnitude in ES8 than in ET8. The functional groups on ET8 and ES8 roots outer layer and inner cells were investigated by Fourier transform infrared spectrometry (FTIR) under Al stress. The FTIR spectra of selected examined areas showed that the characteristic absorption peaks were located at 1692, 2920, and 3380 cm−1. The outer layer cells had stronger peaks than inner cells at wavenumber 1680–1740 cm−1, indicating root outer layer cells contain more carboxyls in both ET8 and ES8. The results demonstrate that Al transverse distribution on plants apical root cross section is likely influenced by functional groups such as negatively charged carboxylic acid.

Identification of Lipopeptide Iturin A Produced by Bacillus amyloliquefaciens NCPSJ7 and Its Antifungal Activities against Fusarium oxysporum f. sp. niveum.

Bacillus amyloliquefaciens NCPSJ7 showed potential fungicidal activities for the effective control of fungal infection. From the PCR test, the key genes (srfAA, sfp, fenD, bmyB, ituD, and ituC) were detected in B. amyloliquefaciens NCPSJ7. These genes were closely related to the lipopeptides (LPs) synthesis. Next, three LPs families were identified with liquid chromatography–mass spectrometry (LC/MS), including iturin A, fengycin A, and surfactin. After purification with C18, the main active antifungal compound was proven to be C14-iturin A by ESI-HRMS, which has significant activities against fungi. These results proved that C14-iturin A played an important role in inhibiting the growth of fungi for B. amyloliquefaciens NCPSJ7. Furthermore, the isolated LP could inhibit mycelial growth and conidia germination at 30 μg/mL. SEM allowed us to observe that mycelial morphology and conidia germination were also affected. The mycelial ultrastructure TEM observations showed that the external electron-dense outer layer cell wall, which mainly consisted of glycoproteins, was affected. Furthermore, swollen mitochondria, enriched glycogen, and increased vacuoles were also found. LP also affected the intact wall and membranes, leading to their increased permeability, which was proved by propidium iodide (PI) staining and conductivity measurements. Meanwhile, the ergosterol, which has an affinity for iturin A, also increased. These results indicated that LP caused fungal dysfunction and membrane permeability increase, leading to fungal inhibition. Identifying and studying LPs is important in exploring the fungicidal activities of B. amyloliquefaciens, which promotes the use of B. amyloliquefaciens NCPSJ7 as a potential candidate for biocontrol.

PcCesA1 is involved in the polar growth, cellulose synthesis, and glycosidic linkage crosslinking in the cell wall of Phytophthora capsici

Phytophthora capsici is a destructive plant pathogen that infects a wide range of hosts worldwide. The P. capsici cell wall, rich in cellulose, is vital for hyphal growth and host interactions. However, the enzymes involved in its synthesis remain largely unelucidated. In the current study, we functionally characterized the cellulose synthase gene PcCesA1, which is highly conserved in Phytophthora. By using CRISPR/Cas9-mediated gene replacement and in situ complementation system, it was found PcCesA1 is essential for the mycelial growth, cystospore germination, and pathogenicity of P. capsici. The normal deposition of newly synthesized cell wall components and the polar growth point formation were disrupted in PcCesA1 knockout mutants, suggesting that PcCesA1 plays an important role in the polar growth of P. capsici. Compared with the wild-type strains, PcCesA1 knockout mutants displayed a thicker inner layer cell wall and were more sensitive to carboxylic acid amide fungicides (CAAs). The contents of the cell wall polysaccharides 1,4-Glc, 1,4,6-Glc, and 1,3,4-Glc were reduced in PcCesA1 knockout mutants, suggesting that PcCesA1 affected cellulose content and glycosidic linkage crosslinking in the cell wall. Our findings demonstrate that PcCesA1 is required for cell wall biogenesis. Therefore, PcCesA1 may be a potential target for Phytophthora disease control.

A Review of Nanotechnology as a Novel Method of Gene Transfer in Plants

Purpose: Nanotechnology has evolved as an effective tool in numerous fields including agriculture, medicine and engineering. Recently it’s potential as an alternative genetic transformation method has been identified. However, a comprehensive understanding over nanoparticles and their behavior in living cells is important to realize the full potential of this technology in biotechnological applications. Therefore, we review the application potential of widely employed nanoparticles in plant transformation here.Literature/Background: Development of new crop varieties with desirable traits via biotechnological applications is a solution for challenges associated with climate change and higher population growth. In such aspects, transformation of plant cells which is known as the process of changing one’s genome by integration exogenous DNA, is an absolute necessity and results far better and improved stable characteristics in original. Rigid and multi layered cell wall impedes penetration of exterior biomolecules and hence causes the transformation process complicated. Even though, numerous conventional methods have been established for plant transformation, lower transformation efficiency, tissue damage and random integration of transgenes warrants the need for novel approaches. In this context, novel techniques have been explored and as a result nanoparticles have been found effective in transformation of protoplasts as well as intact plant cells. Nanoparticles internalized either via endocytosis or direct penetration release transgenes from nanoparticle-DNA complexes and result in transient or stable expression. Nanoparticles ensure higher transformation efficiency, no transgenic silencing and protection of biogenic molecules from degradation by intracellular nucleases.

Mechanical properties of the hollow-wall graphene gyroid lattice

The macroscopic elastic modulus and yield strength of solid-wall nickel gyroids and hollow-wall graphene gyroids of cell size 60 nm are deduced from indentation tests on a thin coating of the gyroids, with suitable interpretation by finite element simulations. The solid-wall nickel gyroids are fabricated by the self-assembly of a triblock copolymer, followed by the chemical vapour deposition of a graphene film onto this catalytic template. The nano-indentation response of the gyroid-based coatings was measured using a Berkovich indenter. In order to interpret the indentation response, two sets of finite element simulations were performed: periodic cell calculations in order to deduce the effective macroscopic properties in terms of the relative density and cell wall properties of the lattice, and then indentation simulations of a continuum with the effective properties of the gyroid. Despite the knockdown in modulus and strength of the graphene gyroid lattice due to waviness of the layered cell walls, the structure remains remarkably strong due to nanoscale size effects.

Fraxinus mandshurica 4-coumarate-CoA ligase 2 enhances drought and osmotic stress tolerance of tobacco by increasing coniferyl alcohol content

4-Coumarate-CoA ligase (4CL) is an important branch point in the phenylpropane pathway and plays important roles in plant growth and development. In this study, the 4CL2 gene from Fraxinus mandshurica (designated Fm4CL2) was identified and isolated. Sequence analysis revealed that Fm4CL2 is a type I 4CL gene involved in lignin biosynthesis. Analysis of cell wall components revealed that Fm4CL2-overexpressing (OE-Fm4CL2) tobacco showed increased lignin content (by 58.9%) and decreased hemicellulose content (by 41.2%). Detection of small-molecule metabolites in the lignin pathway revealed that coumaric acid content decreased by 48% and coniferyl alcohol content increased by 250% compared with the control values. Compared with wild type, OE-Fm4CL2 tobacco showed increased xylem cell layer number (by 120%) and cell wall thickness (by 54.5%). Under osmotic stress, transgenic tobacco showed higher growth than wild-type tobacco. The germination rate of transgenic tobacco was higher than that of wild type. Reactive oxygen species accumulation and malondialdehyde content were significantly lower in transgenic tobacco than in wild type. Under drought, the expression of stress-related genes was higher in 35S–Fm4CL2-infected Fraxinus mandshurica plants than in control plants. These results indicate that Fm4CL2 overexpression can enhance drought and osmotic stress tolerance of plants.

Role of aleurone cell walls in water diffusion and distribution within cereal grains

Wheat (Triticum aestivum L) and barley (Hordeum vulgare L) are two important cereals cultivated worldwide. The effect of aleurone cell wall structure on water diffusion and distribution within wheat and barley grains was evaluated at different relative humidity levels. Time domain nuclear magnetic resonance was used to measure the transverse relaxation time T2 of grains. Two water states were distinguished within grains, namely W1 (lower mobility) and W2 (higher mobility). Grains with thicker aleurone layer cell walls had a higher W2. The water-absorption and desorption rates were mainly determined by the thickness of the aleurone cell walls and decreased with increasing cell wall thickness. The higher W2 values observed in grains with thicker aleurone cell walls with the a water content of 2.0% (w/w) and 12.1% (w/w) were probably related to a higher water motion within the grains, in response to higher porosity. Arabinoxylan and (1,3)(1,4)-β-glucan alternating multilayer films were prepared, each film had 20 layers, one layer was approximately 1 μm thick. The film was used as a model to simulate the aleurone layers. These results show that cell walls of the aleurone layer regulate the diffusion and distribution of water within grains.

Wood Moisture-Induced Swelling at the Cellular Scale—Ab Intra

Wood, a complex hierarchical material, continues to be widely used as a resource to meet humankind’s material needs, in addition to providing inspiration for the development of new biomimetic materials. However, for wood to meet its full potential, researchers must overcome the challenge of understanding its fundamental moisture-related properties across its many levels of hierarchy spanning from the molecular scale up to the bulk wood level. In this perspective, a review of recent research on wood moisture-induced swelling and shrinking is presented from the molecular level to the cellular scale. Numerous aspects of swelling and shrinking in wood remain poorly understood, sub-cellular phenomena in particular, because it can be difficult to study them experimentally. Here, we discuss recent research endeavors at each of the relevant length scales, including the molecular, cellulose elementary fibril, secondary cell wall layer nanostructure, cell wall, cell, and cellular levels. At each length scale, we provide a discussion on the current knowledge and suggestions for future research. The potential impacts of moisture-induced swelling pressures on experimental observations of swelling and shrinking in wood at different length scales are also recognized and discussed.

Anatomy and micromorphology of the endemic Gypsophila osmangaziensis (Caryophyllaceae) and taxonomic contributions for the genus Gypsophila

Gypsophila osmangaziensis, an endemic species from Eskişehir (Turkey) is here studied from the anatomical and micromorphological points of view for the first time. Root, stem and leaf cross-sections as well as seed, leaf and pollen grains surfaces are investigated. Root shows perennial characteristics. Pericycle layer of the stem is composed of sclerenchymatic cells that have 15–17 very thick, layered cell walls. Leaf mesophyll is isobilateral and partly carries druse crystals. Seed surface has an obtuse tubercle. Pollen shape is oblate spheroidal.

Surface deformation during an action potential in pearled cells.

Electric pulses in biological cells (action potentials) have been reported to be accompanied by a propagating cell-surface deformation with a nanoscale amplitude. Typically, this cell surface is covered by external layers of polymer material (extracellular matrix, cell wall material, etc.). It was recently demonstrated in excitable plant cells (Chara braunii) that the rigid external layer (cell wall) hinders the underlying deformation. When the cell membrane was separated from the cell wall by osmosis, a mechanical deformation, in the micrometer range, was observed upon excitation of the cell. The underlying mechanism of this mechanical pulse has, to date, remained elusive. Herein we report that Chara cells can undergo a pearling instability, and when the pearled fragments were excited even larger and more regular cell shape changes were observed (∼10-100μm in amplitude). These transient cellular deformations were captured by a curvature model that is based on three parameters: surface tension, bending rigidity, and pressure difference across the surface. In this paper these parameters are extracted by curve-fitting to the experimental cellular shapes at rest and during excitation. This is a necessary step to identify the mechanical parameters that change during an action potential.

New insight into the disinfection mechanism of Fusarium monoliforme and Aspergillus niger by TiO2 photocatalyst under low intensity UVA light

Titanium dioxide (TiO2) photocatalytic reaction has great potential for the disinfection of harmful pathogens. However, the disinfection mechanisms of TiO2 photocatalysis are not yet well-known for fungi and protozoa. In this work, the photocatalytic disinfection mechanism of Fusarium monoliforme and Aspergillus niger under low intensity UVA light (365nm, <10W/m2) was studied at the ultrastructural level. Photocatalytic treatments showed that the photocatalytic oxidation of 10% TiO2 based paint was efficacious in the complete disinfection of F. monoliforme under low intensity UVA light. No growth of F. monoliforme was observed on agar plate in the subsequent dark. Transmission electron microscopy (TEM) of F. monoliforme exposed to TiO2 photocatalysis treatment showed a distinct damage to electron-dense outer cell wall, but not to an underlying electron-transparent layer cell wall. The TEM image revealed that the UVA-light only did not damage cell wall, cell membrane and cellular organelles. Unlike, A. niger was more sensitive to UVA-light. Serious destructions of cell membrane and cellular organelles were shown in A. niger exposed to UVA-light only and photocatalytic treatments. However, morphological change in A. niger cell wall was only observed in photocatalytic treatment. Changes to the outermost melanin like layer and cell wall of A. niger spore due to photocatalytic treatment were greatly apparent while the intracellular organelles of A. niger spore were not affected. Therefore, regrowth of A. niger on agar plate was expected from the germination of A. niger spore in the subsequent dark. These observations give a better understanding of the photocatalytic disinfection mechanism toward fungi.

Morphogenetic Alterations of Alternaria alternata Exposed to Dicarboximide Fungicide, Iprodione.

Fungicide-resistant Alternaria alternata impede the practical control of the Alternaria diseases in crop fields. This study aimed to investigate cytological fungicide resistance mechanisms of A. alternata against dicarboximide fungicide iprodione. A. alternata isolated from cactus brown spot was cultured on potato-dextrose agar (PDA) with or without iprodione, and the fungal cultures with different growth characteristics from no, initial and full growth were observed by light and electron microscopy. Mycelia began to grow from one day after incubation (DAI) and continued to be in full growth (control-growth, Con-G) on PDA without fungicide, while on PDA with iprodione, no fungal growth (iprodione-no growth, Ipr-N) occurred for the first 3 DAI, but once the initial growth (iprodione-initial growth, Ipr-I) began at 4–5 DAI, the colonies grew and expanded continuously to be in full growth (iprodione-growth, Ipr-G), suggesting Ipr-I may be a turning moment of the morphogenetic changes resisting fungicidal toxicity. Con-G formed multicellular conidia with cell walls and septa and intact dense cytoplasm. In Ipr-N, fungal sporulation was inhibited by forming mostly undeveloped unicellular conidia with degraded and necrotic cytoplasm. However, in Ipr-I, conspicuous cellular changes occurred during sporulation by forming multicellular conidia with double layered (thickened) cell walls and accumulation of proliferated lipid bodies in the conidial cytoplasm, which may inhibit the penetration of the fungicide into conidial cells, reducing fungicide-associated toxicity, and may be utilized as energy and nutritional sources, respectively, for the further fungal growth to form mature colonies as in Ipr-G that formed multicellular conidia with cell walls and intact cytoplasm with lipid bodies as in Con-G.

VARIATIONS IN CELL WALL ULTRASTRUCTURE AND CHEMISTRY IN CELL TYPES OF EARLYWOOD AND LATEWOOD IN ENGLISH OAK (QUERCUS ROBUR)

Although there is considerable information on anatomy and gross chemistry of oak wood, little is known on the ultrastructure and chemistry at the individual cell wall level. In particular, differences in ultrastructure and chemistry within the same cell type between earlywood (EW) and latewood (LW) are poorly understood. This study investigated the ultrastructure and chemistry of (vasicentric) tracheids, vessels, (libriform) fibers and axial/ray parenchyma cells of English oak xylem (Quercus robur L.) using light-, fluorescence- and transmission electron microscopy combined with histo/cytochemistry and immunohisto/ cytochemistry. EW tracheids showed several differences from LW tracheids including thinner cell walls, wider middle lamella cell corner (MLcc) regions and lesser amounts of mannan epitopes. Fibers showed thicker cell walls and higher amounts of mannan epitopes than tracheids. EW vessels were rich in guaiacyl (G) lignin with a characteristic non-layered cell wall organization (absence of S1–3 layers), whereas LW vessels were rich in syringyl (S) lignin with a three layered cell wall structure (S1–3 layers). Formation of a highly lignified and wide protective layer (PL) inside axial/ray parenchyma cells was detected only in EW. Distribution of mannan epitopes varied greatly between cell types and between EW and LW, whereas distribution of xylan epitopes was almost identical in all cell types within a growth ring. Together, this study demonstrates that there are great variations in ultrastructure and chemistry of cell walls within a single growth ring of English oak xylem.

Cellular and Pectin Dynamics during Abscission Zone Development and Ripe Fruit Abscission of the Monocot Oil Palm.

The oil palm (Elaeis guineensis Jacq.) fruit primary abscission zone (AZ) is a multi-cell layered boundary region between the pedicel (P) and mesocarp (M) tissues. To examine the cellular processes that occur during the development and function of the AZ cell layers, we employed multiple histological and immunohistochemical methods combined with confocal, electron and Fourier-transform infrared (FT-IR) microspectroscopy approaches. During early fruit development and differentiation of the AZ, the orientation of cell divisions in the AZ was periclinal compared with anticlinal divisions in the P and M. AZ cell wall width increased earlier during development suggesting cell wall assembly occurred more rapidly in the AZ than the adjacent P and M tissues. The developing fruit AZ contain numerous intra-AZ cell layer plasmodesmata (PD), but very few inter-AZ cell layer PD. In the AZ of ripening fruit, PD were less frequent, wider, and mainly intra-AZ cell layer localized. Furthermore, DAPI staining revealed nuclei are located adjacent to PD and are remarkably aligned within AZ layer cells, and remain aligned and intact after cell separation. The polarized accumulation of ribosomes, rough endoplasmic reticulum, mitochondria, and vesicles suggested active secretion at the tip of AZ cells occurred during development which may contribute to the striated cell wall patterns in the AZ cell layers. AZ cells accumulated intracellular pectin during development, which appear to be released and/or degraded during cell separation. The signal for the JIM5 epitope, that recognizes low methylesterified and un-methylesterified homogalacturonan (HG), increased in the AZ layer cell walls prior to separation and dramatically increased on the separated AZ cell surfaces. Finally, FT-IR microspectroscopy analysis indicated a decrease in methylesterified HG occurred in AZ cell walls during separation, which may partially explain an increase in the JIM5 epitope signal. The results obtained through a multi-imaging approach allow an integrated view of the dynamic developmental processes that occur in a multi-layered boundary AZ and provide evidence for distinct regulatory mechanisms that underlie oil palm fruit AZ development and function.

Induction of Embryogenesis in Brassica Napus Microspores Produces a Callosic Subintinal Layer and Abnormal Cell Walls with Altered Levels of Callose and Cellulose.

The induction of microspore embryogenesis produces dramatic changes in different aspects of the cell physiology and structure. Changes at the cell wall level are among the most intriguing and poorly understood. In this work, we used high pressure freezing and freeze substitution, immunolocalization, confocal, and electron microscopy to analyze the structure and composition of the first cell walls formed during conventional Brassica napus microspore embryogenesis, and in cultures treated to alter the intracellular Ca2+ levels. Our results revealed that one of the first signs of embryogenic commitment is the formation of a callose-rich, cellulose-deficient layer beneath the intine (the subintinal layer), and of irregular, incomplete cell walls. In these events, Ca2+ may have a role. We propose that abnormal cell walls are due to a massive callose synthesis and deposition of excreted cytoplasmic material, and the parallel inhibition of cellulose synthesis. These features were absent in pollen-like structures and in microspore-derived embryos, few days after the end of the heat shock, where abnormal cell walls were no longer produced. Together, our results provide an explanation to a series of relevant aspects of microspore embryogenesis including the role of Ca2+ and the occurrence of abnormal cell walls. In addition, our discovery may be the explanation to why nuclear fusions take place during microspore embryogenesis.

Induction of wound-periderm-like tissue in Kalanchoe pinnata (Lam.) Pers. (Crassulaceae) leaves as a defence response to high UV-B radiation levels.

UV-B radiation can be stressful for plants and cause morphological and biochemical changes. Kalanchoe pinnata is a CAM leaf-succulent species distributed in hot and dry regions, and is rich in flavonoids, which are considered to be protective against UV-B radiation. This study aims to verify if K. pinnata has morphological or anatomical responses as a strategy in response to high UV-B levels. Kalanchoe pinnata plants of the same age were grown under white light (control) or white light plus supplemental UV-B radiation (5 h d(-1)). The plants were treated with the same photoperiod, photosynthetically active radiation, temperature and daily watering system. Fragments of the middle third of the leaf blade and petiole were dehydrated and then embedded in historesin and sectioned in a rotary microtome. Sections were stained with toluidine blue O and mounted in Entellan®. Microchemical analyses by optical microscopy were performed on fresh material with Sudan III, Sudan IV and phloroglucinol, and analysed using fluorescence microscopy. Supplemental UV-B radiation caused leaf curling and the formation of brown areas on the leaves. These brown areas developed into a protective tissue on the adaxial side of the leaf, but only in directly exposed regions. Anatomically, this protective tissue was similar to a wound-periderm, with outer layer cell walls impregnated with suberin and lignin. This is the first report of wound-periderm formation in leaves in response to UV-B radiation. This protective tissue could be important for the survival of the species in desert regions under high UV-B stress conditions.

On radiation damage in FIB-prepared softwood samples measured by scanning X-ray diffraction.

The high flux density encountered in scanning X-ray nanodiffraction experiments can lead to severe radiation damage to biological samples. However, this technique is a suitable tool for investigating samples to high spatial resolution. The layered cell wall structure of softwood tracheids is an interesting system which has been extensively studied using this method. The tracheid cell has a complex geometry, which requires the sample to be prepared by cutting it perpendicularly to the cell wall axis. Focused ion beam (FIB) milling in combination with scanning electron microscopy allows precise alignment and cutting without splintering. Here, results of a scanning X-ray diffraction experiment performed on a biological sample prepared with a focused ion beam of gallium atoms are reported for the first time. It is shown that samples prepared and measured in this way suffer from the incorporation of gallium atoms up to a surprisingly large depth of 1 µm.