Single Cell Wall Research Articles

Isolation and characterization of Methanosphaera sp. ISO3-F5, a member of a novel and widespread species of rumen methanogens growing with methanol plus hydrogen

Rumen methanogens predominantly fall into two physiological groups: hydrogenotrophs which use hydrogen (H2) to reduce carbon dioxide (CO2) to methane (CH4), and methylotrophs which use H2 to reduce methanol and methylamines as substrates for methanogenesis. We used a dilution to extinction approach to isolate two hydrogenotrophic Methanocatella spp. and four cultures of methylotrophic methanogens from sheep rumen contents. Three of the methylotrophs were stable mixed cultures containing methanogens belonging to different lineages of the order Methanomassiliicoccales and one was a pure Methanosphaera culture. Methanosphaera sp. ISO3-F5 has a comparatively large genome (2.68 Mb) comprised of two replicons, a chromosome and a megaplasmid. The genome has an average G + C content of 30.5 % and encodes 2360 putative protein-coding genes. Cells of ISO3-F5 have a spherical shape, 0.6–1.2 µm in diameter, usually occurring in pairs or loose clumps, and have no flagellum. Cells stain Gram positive, have a single thick cell wall and divide by the formation of a cross wall. The optimum temperature for growth was 39°C to 42°C and the optimum pH was 6.7–6.8. Acetate was required for growth, but CH4 was not produced from acetate, formate, ethanol, methylamine, or isopropanol with or without H2/CO2. Volatile fatty acids and rumen fluid were also found to enhance the growth of ISO3-F5, while coenzyme M did not. ISO3-F5 produced CH4 from methanol in the presence of H2 and the genes encoding the necessary methanogenesis pathway have been identified. Based on morphological, physiological, and genomic characteristics, ISO3-F5 is a new species of the genus Methanosphaera. Our study shows that simple isolation methods allowed us to culture diverse and significant members of the rumen methanogen community.

Low oxygen environment effect on the tomato cell wall composition during the fruit ripening process

BackgroundOxygen concentration is a key characteristic of the fruit storage environment determining shelf life and fruit quality. The aim of the work was to identify cell wall components that are related to the response to low oxygen conditions in fruit and to determine the effects of such conditions on the ripening process. Tomato (Solanum lycopersicum) fruits at different stages of the ripening process were stored in an anoxic and hypoxic environment, at 0% and 5% oxygen concentrations, respectively. We used comprehensive and comparative methods: from microscopic immunolabelling and estimation of enzymatic activities to detailed molecular approaches. Changes in the composition of extensin, arabinogalactan proteins, rhamnogalacturonan-I, low methyl-esterified homogalacturonan, and high methyl-esterified homogalacturonan were analysed.ResultsIn-depth molecular analyses showed that low oxygen stress affected the cell wall composition, i.e. changes in protein content, a significantly modified in situ distribution of low methyl-esterified homogalacturonan, appearance of callose deposits, disturbed native activities of β-1,3-glucanase, endo-β-1,4-glucanase, and guaiacol peroxidase (GPX), and disruptions in molecular parameters of single cell wall components. Taken together, the data obtained indicate that less significant changes were observed in fruit in the breaker stage than in the case of the red ripe stage. The first symptoms of changes were noted after 24 h, but only after 72 h, more crucial deviations were visible. The 5% oxygen concentration slows down the ripening process and 0% oxygen accelerates the changes taking place during ripening.ConclusionsThe observed molecular reset occurring in tomato cell walls in hypoxic and anoxic conditions seems to be a result of regulatory and protective mechanisms modulating ripening processes.

A two-photon fluorescent probe for highly selective detection of Cys over GSH and Hcy based on the Michael addition and transcyclization mechanism and its application in bioimaging and protein straining in SDS-PAGE

BackgroundCysteine (Cys), glutathione (GSH), and homocysteine (Hcy), as three major biothiols are involved in a variety of physiological processes and play a crucial role in plant growth. Abnormal levels of Cys can cause plants to fail to grow properly. To date, although a very large number of fluorescent probes have been reported for the detection of biothiols, very few of them can be used for the selective discrimination of Cys from GSH and Hcy due to their structural similarity, and only a few of them can be used for plant imaging. ResultsHere, three fluorescent probes (o-/m-/p-TMA) based on TMN fluorophore and the ortho-/meta-/para-substituted maleimide recognition groups were constructed to investigate the selective response effect of Cys. Compared to the o-/m-TMA, p-TMA can selectively detect Cys over GSH and Hcy with a rapid response time (10 min) and a low detection limit (0.26 μM). The theoretical calculation confirmed that the intermediate p-TMA-Cys-int has shorter interatomic reaction distances (3.827 Å) compared to o-/m-TMA-Cys (5.533/5.287 Å), making it more suitable for further transcyclization reactions. Additionally, p-TMA has been employed for selective tracking of exogenous and endogenous Cys in Arabidopsis thaliana using both single-/two-photon fluorescence imaging. Furthermore, single cell walls produced obvious two-photon fluorescence signals, indicating that p-TMA can be used for high-concentration Cys analysis in single cells. Surprisingly, p-TMA can be used as a fluorescent dye for protein staining in SDS-PAGE with higher sensitivity (7.49 μg/mL) than classical Coomassie brilliant blue (14.11 μg/mL). SignificanceThe outstanding properties of p-TMA make it a promising multifunctional molecular tool for the highly selective detection of Cys over GSH and Hcy in various complex environments, including water solutions, zebrafish, and plants. Additionally, it has the potential to be developed as a fluorescent dye for a simple and fast SDS-PAGE fluorescence staining method.

Elastic properties of honeycombs with spline curve cell walls

In the ideal models, cell walls of honeycomb structures are perfect flat sheets or straight lines (viewed from out-of-plane projection), whereas most of the real honeycomb products contain curved cell walls due to the widely employed manufacturing processes. Moreover, some honeycomb products are made intentionally to have corrugated cell walls for an enhanced out-of-plane stability or an increase of structural stiffness. In the presented study, a relative new modeling method for the nonlinear cell walls of honeycomb structures is used. The method makes use of Bezier spline functions to describe the curved cell walls. Energy method and Castigliano's theorem are used to formulate the force-displacement relationship of a single cell wall, and the homogenized stiffness matrix is derived based on the proper boundary conditions of the cell walls. Analytical and experimental verification shows that the proposed model is very accurate and versatile in predicting the mechanical responses of different honeycomb geometries. Parametric studies are conducted, analytically and numerically, to examine the influence of the spline cell wall geometries on the honeycomb's effective in-plane properties and the out-of-plane stability. The study leads to the recommendation of a cell wall design strategy for maximizing the out-of-plane buckling resistance of honeycomb structures.

Atomic Force Microscopy in the Characterization of the Structure of Cell Wall Components.

Atomic force microscopy (AFM) has broken boundaries in the characterization of the supramolecular architecture of cell wall assemblies and single cell wall polysaccharides at the nanoscale level. Moreover, AFM provides an opportunity to evaluate the mechanical properties of cell wall material which is not possible with any other method. However, in the case of plant tissue, the critical step is a smart sample preparation that should not affect the polysaccharide structure or assembly and on the other hand should consider device limitations, especially scanner ranges. In this chapter, the protocols from the sample preparation, including isolation of cell wall material and extraction of cell wall polysaccharide fractions, through AFM imaging of polysaccharide assemblies and single molecules until an image analysis to obtain quantitative data characterizing the biopolymers are presented.

Adsorption of aflatoxin B1 to corn by-products

The adsorption of aflatoxin B1 (AFB1) to natural fiber materials prepared from corn by-products was investigated in this study. The results showed that corn cob powder (CCP) dose, particle size, time (0.25–24 h), temperature (4, 20, 37, 50 and 100 °C) and pH (2–8), had significant effects on adsorption. The maximum adsorption (98%) was with particles 500–355 µm in size at 20 °C for 8 h, at the dose of 50 mg mL−1. The adsorption fitted pseudo-second-order model and Langmuir isotherm well. Besides, CCP had a higher adsorption capacity to AFB1 than any single cell wall components of corn, which indicated that capillary effect happened in cell wall might be the main reason for adsorption. The results also suggested that CCP could reduce AFB1 content from both liquid and solid food matrixes. Briefly, CCP displayed promising properties that could be developed in nature-based practical applications for food aflatoxin decontamination.

An exhaustive multiple knockout approach to understanding cell wall hydrolase function in Bacillus subtilis.

In order to grow, bacterial cells must both create and break down their cell wall. The enzymes that are responsible for these processes are the target of some of our best antibiotics. Our understanding of the proteins that break down the wall- cell wall hydrolases-has been limited by redundancy among the large number of hydrolases many bacteria contain. To solve this problem, we identified 42 cell wall hydrolases in Bacillus subtilis and created a strain lacking 40 of them. We show that cells can survive using only a single cell wall hydrolase; this means that to understand the growth of B. subtilis in standard laboratory conditions, it is only necessary to study a very limited number of proteins, simplifying the problem substantially. We additionally show that the ∆40 strain is a research tool to characterize hydrolases, using it to identify three "helper" hydrolases that act in certain stress conditions.

Burr formation mechanism and morphological transformation in grinding of nickel-based superalloy honeycomb cores under ice freezing and MQL conditions

Nickel-based superalloy honeycomb core (NBSHC) is extensively used in aerospace field due to the light weight, high specific strength and high temperature resistance. The burrs of NBSHC are extremely prone to be formed during machining and the insufficient welding strength or false welding between NBSHC and skin is generated because of the large or unreasonable burr morphology, resulting in premature failure of the parts. In this work, a novel method of ice freezing combining with minimum quantity lubrication (MQL) was proposed to grind NBSHC to improve the burr morphology and size. The burr formation mechanisms of NBSHC under ice freezing and MQL conditions were revealed by single abrasive particle grinding cell wall and verified by grinding experiments. The grinding force, burr morphology transformation mechanisms and size were investigated in-detail by experiments combining with FEM results. The results indicated that MQL and ice freezing decreased the plastic flow property of material by reducing the temperature and friction of NBSHC, thereby promoting the transformation of burr size from large into micro, and the burr shape from long strip into block. Decreasing grinding depth can also improve the burr morphology by decreasing the volume of plastic flow material. The improving effects of full of ice freezing were better than MQL for NBSHC grinding. The machining efficiency under full of ice combining with MQL was 3 times of that under quasi-dry grinding. These findings are significant for improving the machining quality and exploring the burr morphology transformation of NBSHC.

In situ infrared imaging of the local orientation of cellulose fibrils in plant secondary cell walls.

The mechanical and chemical properties of plant cell walls greatly rely on the supramolecular assembly of cellulose fibrils. To study the local orientation of cellulose in secondary plant cell walls, diffraction limited infrared (IR) micro-spectroscopic mapping experiments were conducted at different orientation of transverse leaf section of the grass Sorghum bicolor with respect to the polarization direction of the IR radiation. Two-dimensional maps, based on polarization-sensitive absorption bands of cellulose were obtained for different polarization angles. They reveal a significant degree of anisotropy of the cellulose macromolecules as well as of other biopolymers in sclerenchyma and xylem regions of the cross section. Quantification of the signals assigned to polarization sensitive vibrational modes allowed to determine the preferential orientation of the sub-micron cellulose fibrils in single cell walls. A sample of crystalline nano-cellulose comprising both a single microcrystal as well as unordered layers of nanocrystals was used for validation of the approach. The results demonstrate that diffraction limited IR micro-spectroscopy can be used to study hierarchically structured materials with complex anisotropic behavior.

Working towards arabinogalactan proteins (AGPs) from fruit: carbohydrate composition and impact on fungal growth

BackgroundArabinogalactan proteins (AGPs) are extracellular matrix constituents involved in plant response to fungal infection. The aim of the current study was to investigate the antifungal effect of AGPs ex situ and to determine the structural features of AGPs that may have an influence on this activity. The features of AGPs isolated from fruit were investigated with molecular tools based on specific monoclonal antibodies recognizing carbohydrate AGP epitopes. The Antifungal (well-diffusion) Susceptibility Test and the Agar Invasion Test were used to assess the impact of AGPs on Penicillium notatum culture.ResultsThe results definitely ruled out the influence of AGPs on fungal growth. The immunochemical analyses revealed that AGPs consist mainly of carbohydrate chains composed of β-linked glucuronosyl residues recognized by LM2 and GlcA-β(1 → 3)-GalA-α(1 → 2) Rha recognized by JIM13, which do not have the same functional properties outside the plant cell in in vitro experimental conditions.ConclusionsThe action of a single cell wall component does not elicit any influence ex situ. The extensive accumulation of glycan chains of AGPs in infected tissue as a result of a complex mechanism occurring in the cell wall emphasizes the importance of dependencies between particular components of the extracellular matrix in response to fungal attack.

Effect of foam filling on the energy absorption behaviour of flax/polylactic acid composite interlocking sandwich structures

A novel double cell wall interlocking square core was fabricated from flax fibre reinforced polylactic acid composite. Initially, a series of square core panels with cell sizes of 20, 30, and 40 mm were manufactured using hot press machine and later fabricated using a simple slotting technique. Then, series of quasi-static compression test loadings with a crosshead displacement rate of 2 mm/min were carried out on empty and filled-foam core structure with different densities. Here, the effect of using double cell walls, different cell sizes and foam-filled core structure were investigated. Based on the results, the compressive strength and specific energy absorption (SEA) increased by up to 3.45–126% and 3.75–64% with respect to empty core structure. It was also demonstrated that the higher the foam density filled in a smaller cell size core structure, the greater the effect on its strength and SEA. In addition, the variation of the cell core sizes was more pronounced in the case of the empty core structure compared to their foam-filled core counterparts. Finally, the current results were found to be between 7 and 10 times higher than those of single cell walls reported in the literature in terms of their strength and SEA.

Subcellular coordination of plant cell wall synthesis.

Organelles of the plant cell cooperate to synthesize and secrete a strong yet flexible polysaccharide-based extracellular matrix: the cell wall. Cell wall composition varies among plant species, across cell types within a plant, within different regions of a single cell wall, and in response to intrinsic or extrinsic signals. This diversity in cell wall makeup is underpinned by common cellular mechanisms for cell wall production. Cellulose synthase complexes function at the plasma membrane and deposit their product into the cell wall. Matrix polysaccharides are synthesized by a multitude of glycosyltransferases in hundreds of mobile Golgi stacks, and an extensive set of vesicle trafficking proteins govern secretion to the cell wall. In this review, we discuss the different subcellular locations at which cell wall synthesis occurs, review the molecular mechanisms that control cell wall biosynthesis, and examine how these are regulated in response to different perturbations to maintain cell wall homeostasis.

Arabinoxylan, β-glucan and pectin in barley and malt endosperm cell walls: a microstructure study using CLSM and cryo-SEM.

The architecture of endosperm cell walls in Hordeum vulgare (barley) differs remarkably from that of other grass species and is affected by germination or malting. Here, the cell wall microstructure is investigated using (bio)chemical analyses, cryogenic scanning electron microscopy (cryo-SEM) and confocal laser scanning microscopy (CLSM) as the main techniques. The relative proportions of β-glucan, arabinoxylan and pectin in cell walls were 61, 34 and 5%, respectively. The average thickness of a single endosperm cell wall was 0.30µm, as estimated by the cryo-SEM analysis of barley seeds, which was reduced to 0.16µm after malting. After fluorescent staining, 3D confocal multiphoton microscopy (multiphoton CLSM) imaging revealed the complex cell wall architecture. The endosperm cell wall is composed of a structure in which arabinoxylan and pectin are colocalized on the outside, with β-glucan depositions on the inside. During germination, arabinoxylan and β-glucan are hydrolysed, but unlike β-glucan, arabinoxylan remains present in defined cell walls in malt. Integrating the results, an enhanced model for the endosperm cell walls in barley is proposed.

Catalytic Decomposition of the Oleaginous Yeast Cutaneotrichosporon Oleaginosus and Subsequent Biocatalytic Conversion of Liberated Free Fatty Acids

A single step catalytic cell wall lysis and triglyceride hydrolysis combined with the enzymatic conversion of lipids using the oleaginous yeast Cutaneotrichosporon oleaginosus (ATCC 20509) as a mod...

Titan cells formation in Cryptococcus neoformans is finely tuned by environmental conditions and modulated by positive and negative genetic regulators.

The pathogenic fungus Cryptococcus neoformans exhibits morphological changes in cell size during lung infection, producing both typical size 5 to 7 μm cells and large titan cells (> 10 μm and up to 100 μm). We found and optimized in vitro conditions that produce titan cells in order to identify the ancestry of titan cells, the environmental determinants, and the key gene regulators of titan cell formation. Titan cells generated in vitro harbor the main characteristics of titan cells produced in vivo including their large cell size (>10 μm), polyploidy with a single nucleus, large vacuole, dense capsule, and thick cell wall. Here we show titan cells derived from the enlargement of progenitor cells in the population independent of yeast growth rate. Change in the incubation medium, hypoxia, nutrient starvation and low pH were the main factors that trigger titan cell formation, while quorum sensing factors like the initial inoculum concentration, pantothenic acid, and the quorum sensing peptide Qsp1p also impacted titan cell formation. Inhibition of ergosterol, protein and nucleic acid biosynthesis altered titan cell formation, as did serum, phospholipids and anti-capsular antibodies in our settings. We explored genetic factors important for titan cell formation using three approaches. Using H99-derivative strains with natural genetic differences, we showed that titan cell formation was dependent on LMP1 and SGF29 genes. By screening a gene deletion collection, we also confirmed that GPR4/5-RIM101, and CAC1 genes were required to generate titan cells and that the PKR1, TSP2, USV101 genes negatively regulated titan cell formation. Furthermore, analysis of spontaneous Pkr1 loss-of-function clinical isolates confirmed the important role of the Pkr1 protein as a negative regulator of titan cell formation. Through development of a standardized and robust in vitro assay, our results provide new insights into titan cell biogenesis with the identification of multiple important factors/pathways.

Inhomogeneity of Cellulose Microfibril Assembly in Plant Cell Walls Revealed with Sum Frequency Generation Microscopy.

Sum frequency generation (SFG) vibrational spectroscopy can selectively detect and analyze noncentrosymmetric components interspersed in amorphous matrices; this principle has been used for studies of nanoscale structure and mesoscale assembly of cellulose in plant cell walls. However, the spectral information averaged over a large area or volume cannot provide regiospecific or tissue-specific information of different cells in plants. This study demonstrates spatially resolved SFG analysis and imaging by combining a broad-band SFG spectroscopy system with an optical microscope. The system was designed to irradiate both narrow-band 800 nm and broad-band tunable IR beams through a single reflective objective lens, but from opposite sides of the surface normal direction of the sample. The developed technique was used to reveal inhomogeneous distributions of cellulose microfibrils within single cell walls, such as cotton fibers and onion epidermis as well as among different tissues in Arabidopsis inflorescence stems and bamboo culms. SFG microscopy can be used for vibrational spectroscopic imaging of other biological systems in complement to conventional Fourier transform infrared spectroscopy and confocal Raman microscopy.

Wood nails to fix softwoods: characterization of structural deformation and lignin modification

Softwoods can be nailed with high density solid wood like Santos Rosewood (Machaerium scleroxylon) or nails made of densified laminated beech wood composite by hammering or shooting, for example with air nail guns, without pre-drilling. Shooting with about 30 m/s causes friction between nail surface and softwood matrix, heating up the interface to temperatures high enough to soften lignin. Re-condensation of softened lignin causes wood welding which doubles pull out strength in comparison to wood nails fixed without wood welding. Changes in lignin UV-absorption within the single cell wall layer and deformation of main anatomical features of spruce wood tissue are characterized by scanning UV microspectrophotometry (UMSP) whereas structural deformations of spruce wood tissue are revealed by X-ray micro-computed tomography (µ-CT).

Comparison of the mechanical characteristics of fibers and cell walls from moso bamboo and wood

Bamboo and wood fibers are important raw materials for pulp and papermaking, as well as fiber-reinforced composites. The mechanical properties of single fibers and the cell walls of moso bamboo (Phyllostachys heterocycla), Masson pine (Pinus massoniana), and Chinese fir (Cunninghamia lanceolata) were tested via single fiber tensile test and nanoindentation; their fracture characteristics were also compared. The single fibers and cell walls of moso bamboo had superior mechanical properties compared with those of Masson pine and Chinese fir. The bamboo fibers exhibited high strength, high elasticity, and superior ductility. The results indicated that the differences between the mechanical properties of the fiber cells and cell walls of moso bamboo and those of wood were largely dependent upon cell shape and structure.

Effects of Biodegradation on the Structure and Properties of Windmill Palm (Trachycarpus fortunei) Fibers Using Different Chemical Treatments.

In this work, windmill palm fiber (WPF), alkali-treated fiber (AF) without hemicellulose and bleached fiber (BF) without lignin were prepared and buried in soil for 30, 60 and 90 days. The surface morphology, chemical composition, crystallinity degree, mechanical properties, and residual mass rate of the samples, before and after biodegradation, were investigated. According to the results, soil burial degradation can remove the parenchyma cells and silica-bodies of WPF and deplete droplets containing the lignin of alkali-treated fiber after it has been buried for 30 days (AF30), and degradation of the single fiber cell wall of bleached fiber after it has been buried for 30 days (BF30). Buried in natural soil, lignin has a slower degradation rate than that of hemicellulose. WPF showed no significant differences in tensile strength after burial in soil for 90 days, because of the integrity fiber structure decreased the biodegradation. The most serious decrease, about 43%, in tensile strength occurred in AF after it had been buried for 90 days (BF90). This basic knowledge may be helpful for windmill palm fiber applications, especially for biodegradable composites.

Immunolocalization of pectin and hemicellulose epitopes in the phloem of Norway spruce and Scots pine

Key messageDistribution patterns of pectin and hemicellulose epitopes in the phloem of Pinaceae conifers differ between cell types including sieve cells, axial/ray parenchyma cells and stone cells, which is more prominent in seedlings than mature trees.Although there is considerable information on the gross chemistry of conifer bark, little is known on the chemistry of secondary phloem at the individual cell level. This study investigated distribution of pectins and hemicelluloses in the phloem of two conifer species (Norway spruce and Scots pine) at an individual cell wall level using nine monoclonal antibodies specific for pectin and hemicellulose epitopes combined with immunofluorescence and TEM immunogold labeling. Differences in phloem cell wall chemistry between juvenile (seedlings) and mature conifer trees were also examined. The two conifer species showed qualitatively similar distribution patterns of epitopes in sieve- and (axial/ray) parenchyma cells, irrespective of seedlings and mature trees. Sieve- and parenchyma cell walls showed the presence of rhamnogalacturonan-I (RG-I), homogalacturonan (HG) and xyloglucan epitopes, but revealed the absence of heteroxylan epitopes. Heteromannan epitopes were only detected in sieve cell walls, showing a chemical difference between sieve- and parenchyma cells. In contrast to qualitative similarity, there were several quantitative differences of epitope localization in sieve- and parenchyma cells between the two conifer species, indicating variations in the chemical structure and/or the amount of pectins and hemicelluloses between the two conifer species. These differences were more significant in seedlings than mature trees. Immunogold labeling of Norway spruce seedlings further indicated the possibility of chemical variations between cell wall regions within a single sieve cell wall. Phloem stone cells detected in mature Norway spruce showed the presence of heteromannans/heteroxylans and RG-I/HG/xyloglucans in secondary cell wall and middle lamellae, respectively.