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.

Immunohistochemical Detection of the Wall Components on the Example of Shoot Apical Meristem of Fagopyrum esculentum and Fagopyrum tataricum.

Immunohistochemistry is a method that allows the detection of individual components of cell walls in an extremely precise way at the level of a single cell and wall domains. The cell wall antibodies detect specific epitopes of pectins, arabinogalactan proteins (AGP), hemicelluloses, and extensins. The presented method visualization of the selected pectic and AGP epitopes using antibodies directed to wall components is described. The method of the analysis of the chemical composition of the wall is present on the example of the shoot apical meristems of Fagopurum esculentum and Fagopyrum tataricum. Recommended protocols for immunostaining and examination on fluorescence microscopy level are presented.

Role of microfibril angle in molecular deformation of cellulose fibrils in Pinus massoniana compression wood and opposite wood studied by in-situ WAXS.

Upon tensile stress, the spiral cellulose fibrils in wood cell walls rotate like springs with decreasing microfibril angle (MFA), and the cellulose molecules elongate in the chain direction. Compression wood with high MFA and opposite wood with low MFA were comparatively studied by in-situ tensile tests combined with synchrotron radiation WAXS in the present study. FTIR spectroscopy revealed that compression wood had a higher lignin content and fewer acetyl groups. For both types of wood, the lattice spacing d004 increased and the MFA decreased gradually with the increase of tensile stress. At stresses beyond the yield point, cellulose lattice strain depended linearly on macroscopic stress, while the MFA depended linearly on macroscopic strain. The deformation mechanisms of compression wood and opposite wood are not essentially different but differ in their deformation behavior. Specifically, the contribution ratio of lattice strain and cellulose fibril reorientation to macroscopic strain was 0.25 and 0.53 for compression wood, and 0.40 and 0.33 for opposite wood, respectively. Due to the geometric effects of MFA, a greater contribution of cellulose fibril reorientation to the macroscopic deformation was detected in compression wood than in opposite wood.

Yeast cell wall capsules for delivery of oat biomarker avenanthramide-C.

Avenanthramide-C (AVN-C) is the biomarker for oat with a variety of physiological functions, whereas its application is constrained by low stability and bioavailability. Avenanthramide-C is the biomarker for oat with a variety of physiological functions, whereas its application is constrained by low stability and bioavailability. This study evaluated the potential of yeast cell (YC) and yeast cell wall (YCW) capsules as delivery systems for stabilizing AVN-C. It was observed that these yeast capsules possessed the ellipsoidal morphology and intact structure without visual pores. Additionally, the YCW capsules exhibited higher encapsulation and loading capacity due to the large internal space. The interaction of yeast capsules with AVN-C involved the hydrophobic interactions and hydrogen bonding. Moreover, the loading of AVN-C induced high hydrophobicity inside the yeast capsules, which helped to protect AVN-C against degradation and release AVN-C in a slow and sustained manner in the simulated gastrointestinal tract. The YCW capsules have potential as controlled delivery system for AVN-C, which could be further used as a nutraceutical and added to functional foods.

Proteomic Analysis of Cell Wall Proteins with Various Linkages in Fusarium graminearum.

The fungal cell wall, primarily comprising a glucan-chitin matrix and cell wall proteins (CWPs), serves as a key mediator for fungal interactions with the environment and plays a pivotal role in virulence. In this study, we employed a comprehensive proteomics approach to analyze the CWPs in the plant pathogenic fungus Fusarium graminearum. Our methodology successfully extracted and identified 1373 CWPs, highlighting their complex linkages, including noncovalent bonds, disulfide bridges, alkali-sensitive linkages, and glycosylphosphatidylinositol (GPI) anchors. A significant subset of these proteins, enriched in Gene Ontology terms, suggest multifunctional roles of CWPs. Through the integration of transcriptomic and proteomic data, we observed differential expression patterns of CWPs across developmental stages. Specifically, we focused on two genes, Fca7 and Cpd1, which were upregulated in planta, and confirmed their localization predominantly outside the plasma membrane, primarily in the cell wall and periplasmic space. The disruption of FCA7 reduced virulence on wheat, aligning with previous findings and underscoring its significance. Overall, our findings offer a comprehensive proteomic profile of CWPs in F. graminearum, laying the groundwork for a deeper understanding of their roles in the development and interactions with host plants.

Structure of rhamnoglucan, an unexpected alkali-stable polysaccharide extracted from Streptococcus mutans cell wall.

This study identified a rhamnose-containing cell wall polysaccharide (RhaCWP) in an alkaline extract prepared to analyze intracellular polysaccharides (IPS) from Streptococcus mutans biofilm. IPS was an 1,4-α-D-glucan with branchpoints introduced by 1,6-α-glucan while RhaCWP presented 1,2-α-L-and 1,3-α-L rhamnose backbone and side chains connected by 1,2-α-D-glucans, as identified by nuclear magnetic resonance (NMR) spectroscopy and methylation analyses. The MW of IPS and RhaCWP was 11,298 Da, as determined by diffusion-ordered NMR spectroscopy. Therefore, this study analyzed the chemical structure of RhaCWP and IPS from biofilm in a single fraction prepared via a convenient hot-alkali extraction method. This method could be a feasible approach to obtain such molecules and improve the comprehension of the structure-function relationships in polymers from S. mutans in future studies.

Synthesis of a unique mannose α-1-phosphate side chain moiety found in Candida auris cell wall mannan.

Candida auris is an emerging fungal pathogen that has become a world-wide public health threat. While there have been numerous studies into the nature, composition and structure of the cell wall of Candida albicans and other Candida species, much less is known about the C. auris cell wall. We have shown that C. auris cell wall mannan contains a unique phosphomannan structure which distinguishes C. auris mannan from the mannans found in other fungal species. Specifically, C. auris exhibits two unique acid-labile mannose α-1-phosphate (Manα1PO4) sidechains that are absent in other fungal mannans and fungal pathogens. This unique mannan structural feature presents an opportunity for the development of vaccines, therapeutics, diagnostic tools and/or research reagents that target C. auris. Herein, we describe the successful synthesis and structural characterization of a Manα1PO4-containing disaccharide moiety that mimics the phosphomannan found in C. auris. Additionally, we present evidence that the synthetic Manα1PO4 glycomimetic is specifically recognized and bound by cell surface pattern recognition receptors, i.e. rhDectin-2, rhMannose receptor and rhMincle, that are known to play important roles in the innate immune response to C. auris as well as other fungal pathogens. The synthesis of the Manα1PO4 glycomimetic may represent an important starting point in the development of vaccines, therapeutics, diagnostics and research reagents which target a number of C. auris clinical strains. In addition, these data provide new insights and understanding into the structural biology of this unique fungal pathogen.

Saccharomyces cerevisiae CellWall Remodeling in the Absence of Knr4 and Kre6 Revealed by Nano-FourierTransform Infrared Spectroscopy.

The cell wall integrity (CWI) signaling pathway regulates yeast cell wall biosynthesis, cell division, and responses to external stress. The cell wall, comprised of a dense network of chitin, ß-1,3- and ß-1,6- glucans, and mannoproteins, is very thin, <100 nm. Alterations in cell wall composition may activate the CWI pathway. Saccharomyces cerevisiae, a model yeast, was used to study the role of individual wall components in altering the structure and biophysical properties of the yeast cell wall. Near-field Fourier transform infrared spectroscopy (nano-FT-IR) was used for the first direct, spectrochemical identification of cell wall composition in a background (wild-type) strain and two deletion mutants from the yeast knock-out collection: kre6Δ and knr4Δ. Killer toxin resistant 6 (Kre6) is an integral membrane protein required for biosynthesis of ß-1,6-glucan, while Knr4 is a cell signaling protein involved in the control of cell wall biosynthesis, in particular, biosynthesis and deposition of chitin. Complementary spectral data were obtained with far-field (FF)-FT-IR, in transmission, and with attenuated total reflectance (ATR) spectromicroscopy with 3-10 µm wavelength-dependent spatial resolution. The FF-FT-IR spectra of cells and spectra of isolated cell wall components showed that components of the cell body dominated transmission spectra and were still evident in ATR spectra. In contrast, the nano-FT-IR at ∼25 nm spatial resolution could be used to characterize the yeast wall chemical structure. Our results show that the ß-1,6-glucan content is decreased in kre6Δ, while all glucan content is decreased in the knr4Δ cell wall. The latter may be thinner than in wild type, since not only are mannan and chitin detectable by nano-FT-IR, but also lipid membranes and protein, indicative of cell interior.

Streamlining assays of glycosyltransferases activity using in vitro GT-array (i-GT-ray) platform: Application to family GT37 fucosyltransferases.

Numerous putative glycosyltransferases (GTs) have been identified using bioinformatic approaches. However, demonstrating the activity of these GTs remains a challenge. Here, we describe the development of a rapid in vitro GT-array screening platform for activity of GTs. GT-arrays are generated by cell-free in vitro protein synthesis and binding using microplates precoated with a N-terminal Halo- or a C-terminal GST-tagged GT-encoding plasmid DNA and a capture antibody. These arrays are then used for screening of transferase activities and the reactions are monitored by a luminescence GLO assay. The products formed by these reactions can be analyzed directly from the microplates by mass spectrometry. Using this platform, a total of 280 assays were performed to screen 22 putative fucosyltransferases (FUTs) from family GT37 (seven from Arabidopsis and 15 from rice) for activity toward five acceptors: non-fucosylated tamarind xyloglucan (TXyG), arabinotriose (Ara3), non-fucosylated rhamnogalacturonan I (RG-I), and RG-II from the mur1-1 Arabidopsis mutant, and the celery RG-II monomer lacking Arap and MeFuc of chain B and l-Gal of chain A. Our screen showed that AtFUT2, AtFUT5, and AtFUT10 have activity toward RG-I, while AtFUT8 was active on RG-II. Five rice OsFUTs have XyG-FUT activity and four rice OsFUTs have activity toward Ara3. None of the putative OsFUTs were active on the RG-I and RG-II. However, promiscuity toward acceptors was observed for several FUTs. These findings extend our knowledge of cell wall polysaccharide fucosylation in plants. We believe that in vitro GT-array platform provides a valuable tool for cell wall biochemistry and other research fields.

Degradation of Cell Wall Polysaccharides during Traditional and Tank Fermentation of Chinese Liupao Tea.

The increase of polysaccharides in the dark tea pile process is thought to be connected to the cell wall polysaccharides' breakdown. However, the relationship between tea polysaccharides (TPSs) and tea cell wall polysaccharides has not been further explored. In this study, the structural changes in the cell wall polysaccharides [e.g., cellulose, hemicellulose (HC), and pectin] in Liupao tea were characterized before and after traditional fermentation and tank fermentation. Additionally, the degradation mechanism of tea cell wall polysaccharides during fermentation was assessed. The results showed that cellulose crystallinity decreased by 11.9-49.6% after fermentation. The molar ratio of monosaccharides, such as arabinose, rhamnose, and glucose in HC, was significantly reduced, and the molecular weight decreased. The esterification degree and linearity of water-soluble pectin (WSP) were reduced. TPS content increases during pile fermentation, which may be due to HC degradation and the increase in WSP caused by cell wall structure damage. Microorganisms were shown to be closely associated with the degradation of cell wall polysaccharides during fermentation according to correlation analyses. Traditional fermentation had a greater effect on the cellulose structure, while tank fermentation had a more noticeable impact on HC and WSP.

Pectic-AGP is a major form of Arabidopsis AGPs.

As a key component in cell walls of numerous organisms ranging from green algae to higher plants, AGPs play principal roles in many biological processes such as cell-cell adhesion and regulating Ca2+ signaling pathway as a Ca2+-capacitor. Consistently, AGP structures vary from species to species and from tissue to tissue. To understand the functions of AGPs, it is vital to know their structural differences relative to their location in the plant. Thus, AGPs were purified from different Arabidopsis tissues. Analyses of these AGPs demonstrated that the AGPs comprised covalently linked pectin and AGP, referred to as pectic-AGPs. Importantly, these pectic-AGPs were glycosylated with a remarkable variety of polysaccharides including homogalacturonan, rhamnogalacturonan-I, and type II arabinogalactan at different ratios and lengths. This result not only suggests that pectic-AGP is a major form of Arabidopsis AGPs, but also supports AGPs serve as crosslinkers covalently connecting pectins with structures tailored for tissue-specific functions.

The basic leucine zipper transcription factor MeaB is critical for biofilm formation, cell wall integrity, and virulence in Aspergillus fumigatus.

The regulation of fungal cell wall biosynthesis is crucial for cell wall integrity maintenance and directly impacts fungal pathogen virulence. Although numerous genes are involved in fungal cell wall polysaccharide biosynthesis through multiple pathways, the underlying regulatory mechanism is still not fully understood. In this study, we identified and functionally characterized a direct downstream target of SomA, the basic-region leucine zipper transcription factor MeaB, playing a certain role in Aspergillus fumigatus cell wall integrity. Loss of meaB reduces hyphal growth, causes severe defects in galactosaminogalactan-mediated biofilm formation, and attenuates virulence in a Galleria mellonella infection model. Furthermore, the meaB null mutant strain exhibited hypersensitivity to cell wall-perturbing agents and significantly alters the cell wall structure. Transcriptional profile analysis revealed that MeaB positively regulates the expression of the galactosaminogalactan biosynthesis and ß-1,3-glucanosyltransferase genes uge3, agd3, and sph3 and gel1, gel5, and gel7, respectively, as well as genes involved in amino sugar and nucleotide sugar metabolism. Further study demonstrated that MeaB could respond to cell wall stress and contribute to the proper expression of mitogen-activated protein kinase genes mpkA and mpkC in the presence of different concentrations of congo red. In conclusion, A. fumigatus MeaB plays a critical role in cell wall integrity by governing the expression of genes encoding cell wall-related proteins, thus impacting the virulence of this fungus.IMPORTANCEAspergillus fumigatus is a common opportunistic mold that causes life-threatening infections in immunosuppressed patients. The fungal cell wall is a complex and dynamic organelle essential for the development of pathogenic fungi. Genes involved in cell wall polysaccharide biosynthesis and remodeling are crucial for fungal pathogen virulence. However, the potential regulatory mechanism for cell wall integrity remains to be fully defined in A. fumigatus. In the present study, we identify basic-region leucine zipper transcription factor MeaB as an important regulator of cell wall galactosaminogalactan biosynthesis and ß-1,3-glucan remodeling that consequently impacts stress response and virulence of fungal pathogens. Thus, we illuminate a mechanism of transcriptional control fungal cell wall polysaccharide biosynthesis and stress response. As these cell wall components are promising therapeutic targets for fungal infections, understanding the regulatory mechanism of such polysaccharides will provide new therapeutic opportunities.

The legacy of terrestrial plant evolution on cell wall fine structure.

The evolution of land flora was an epochal event in the history of planet Earth. The success of plants, and especially flowering plants, in colonizing all but the most hostile environments required multiple mechanisms of adaptation. The mainly polysaccharide-based cell walls of flowering plants, which are indispensable for water transport and structural support, are one of the most important adaptations to life on land. Thus, development of vasculature is regarded as a seminal event in cell wall evolution, but the impact of further refinements and diversification of cell wall compositions and architectures on radiation of flowering plant families is less well understood. We approached this from a glyco-profiling perspective and, using carbohydrate microarrays and monoclonal antibodies, studied the cell walls of 287 plant species selected to represent important evolutionary dichotomies and adaptation to a variety of habitats. The results support the conclusion that radiation of flowering plant families was indeed accompanied by changes in cell wall fine structure and that these changes can obscure earlier evolutionary events. Convergent cell wall adaptations identified by our analyses do not appear to be associated with plants with similar lifestyles but that are taxonomically distantly related. We conclude that cell wall structure is linked to phylogeny more strongly than to habitat or lifestyle and propose that there are many approaches of adaptation to any given ecological niche.

Effect of Moisture Content on Ion Diffusion and Glass Transition Temperature in Wood Cell Walls.

Understanding and controlling the diffusion of ions and chemicals within the secondary plant cell walls are pivotal in various applications of biomasses. Recent studies have shown that inorganic ion diffusion through secondary cell walls is controlled by a moisture-induced glass transition in amorphous polysaccharides, including amorphous cellulose and hemicelluloses. Understanding the diffusion of ions in these structures has been the subject of numerous recent experiments; however, a deep understanding of the underlying mechanisms of interactions between ion atoms and water/hemicellulose molecules is still lacking. This study uses molecular dynamics simulations to elucidate the diffusion mechanisms of potassium and chloride ions in the cell walls under varying moisture content. The results reveal that a higher moisture content leads to the formation of solvent layers around the ions and reduces the charge interaction between the functional groups of wood polymers and ions. Hence, a higher moisture content results in an improved diffusion rate of ions within the domain. The simulation results also show that higher moisture content lowers the glass transition temperature, promoting diffusion of ions in the system. In contrast, increases in the ion concentration increase the glass transition temperature of the system and degrade the diffusion of ions in the system.

Hydration and mechanical properties of arabinoxylan, (1,3;1,4)-ß-glucan, and cellulose multilayer films simulating the cell wall of wheat endosperm.

The cell walls of wheat endosperm, which play a pivotal role in seed germination, exhibit a laminated structure primarily composed of polysaccharides. In this study, composite multilayer films were prepared using arabinoxylan (AX), (1,3;1,4)-ß-D-glucan (MLG), and cellulose nanofibers (CNFs), and the effect of polymer blend structure on cell wall hydration and mechanical properties was investigated. Atomic force microscopy and X-ray diffraction indicated that the network structure of MLG/CNF exhibits a higher degree of continuity and uniformity compared to that of AX/CNF. Mechanically, the extensive linkages between MLG and CNFs chains enhance the mechanical properties of the films. Moreover, water diffusion experiments and TD-NMR analysis revealed that water molecules diffuse faster in the network structure formed by AX. We propose a structural model of the endosperm cell wall, in which the CNFs polymer blend coated with MLG serves as the framework, and the AX network fills the gaps between them, providing diffusion channels for water molecules.

The nonlinear mechanics of highly extensible plant epidermal cell walls.

Plant epidermal cell walls maintain the mechanical integrity of plants and restrict organ growth. Mechanical analyses can give insights into wall structure and are inputs for mechanobiology models of plant growth. To better understand the intrinsic mechanics of epidermal cell walls and how they may accommodate large deformations during growth, we analyzed a geometrically simple material, onion epidermal strips consisting of only the outer (periclinal) cell wall, ~7 µm thick. With uniaxial stretching by >40%, the wall showed complex three-phase stress-strain responses while cyclic stretching revealed reversible and irreversible deformations and elastic hysteresis. Stretching at varying strain rates and temperatures indicated the wall behaved more like a network of flexible cellulose fibers capable of sliding than a viscoelastic composite with pectin viscosity. We developed an analytic framework to quantify nonlinear wall mechanics in terms of stiffness, deformation, and energy dissipation, finding that the wall stretches by combined elastic and plastic deformation without compromising its stiffness. We also analyzed mechanical changes in slightly dehydrated walls. Their extension became stiffer and more irreversible, highlighting the influence of water on cellulose stiffness and sliding. This study offers insights into the structure and deformation modes of primary cell walls and presents a framework that is also applicable to tissues and whole organs.

Fractionation and characterization of cell wall polysaccharides from coffee (Coffea arabica L.) pulp.

Cell wall polysaccharides were isolated by sequential extractions from coffee pulp, the main solid waste from coffee processing. Extractions were conducted with distilled water at room and boiling temperatures, 0.5 % ammonium oxalate and 0.05 M Na2CO3 to obtain pectic fractions. Hemicelluloses were extracted by using 2 M and 4 M NaOH. The composition of the hemicellulose fractions suggested the presence of xyloglucans, galactomannans and arabinogalactan-proteins (AGPs). The main part of the cell wall polysaccharides recovered from coffee pulp were pectins branched with arabinogalactans. Coffee pulp pectic fractions were low-methoxylated with various amounts of protein (0.5-8.4 %) and phenolics (0.7-8.5 %). Detection at 280 nm in the HPSEC analyses and radial gel diffusion assay using Yariv reagent indicated the presence of AGPs in most of these fractions. NMR analyses of chelating agent (CSP) and dialyzed water (WSPD) extracted pectins were carried out. The results demonstrated that CSP contains only AG I. On the other hand, AG I and AG II are present in WSPD, probably covalently linked to the pectic portion. Comparison with the literature indicated similarities between the cell wall polysaccharides from coffee pulp and green coffee beans.

Insights into Peptidoglycan-Targeting Radiotracers for Imaging Bacterial Infections: Updates, Challenges, and Future Perspectives.

The unique structural architecture of the peptidoglycan allows for the stratification of bacteria as either Gram-negative or Gram-positive, which makes bacterial cells distinguishable from mammalian cells. This classification has received attention as a potential target for diagnostic and therapeutic purposes. Bacteria's ability to metabolically integrate peptidoglycan precursors during cell wall biosynthesis and recycling offers an opportunity to target and image pathogens in their biological state. This Review explores the peptidoglycan biosynthesis for bacteria-specific targeting for infection imaging. Current and potential radiolabeled peptidoglycan precursors for bacterial infection imaging, their development status, and their performance in vitro and/or in vivo are highlighted. We conclude by providing our thoughts on how to shape this area of research for future clinical translation.

Recent advances in the biosynthesis of fungal glucan structural diversity.

Glucans are the most abundant class of macromolecule polymers in fungi, which are commonly found in Ascomycota and Basidiomycota. Fungal glucans are not only essential for cell integrity and function but also crucial for the immense industrial interest in high value applications. They present a variety of structural characteristics at the nanoscale due to the high regulation of genes and the involvement of stochastic processes in synthesis. However, although recent findings have demonstrated the genes of glucans synthesis are relatively conserved across diverse fungi, the formation and organization of diverse glucan structures is still unclear in fungi. Here, we summarize the structural features of fungal glucans and the recent developments in the mechanisms of glucans biosynthesis. Furthermore, we propose the engineering strategies of targeted glucan synthesis and point out the remaining challenges in the synthetic process. Understanding the synthesis process of diverse glucans is necessary for tailoring high value glucan towards specific applications. This engineering strategy contributes to enable the sustainable and efficient production of glucan diversity.

Side chain of confined xylan affects cellulose integrity leading to bending stem with reduced mechanical strength in ornamental plants.

The stem support for fresh-cut flowers exerts a profound influence on the display of their blossoms. During vase insertion, bending stems significantly affect the ornamental value, but much remains unclear about the underlying reasons. In this study, six pairs of ornamental plants were screened for the contrast of bending and straight stems. The bending stems have weakened mechanical force and biomass recalcitrance compared with the straight ones. Meanwhile, cells in the bending stems became more loosely packed, along with a decrease in cell wall thickness and cellulose levels. Furthermore, wall properties characterizations show bending stems have decreased lignocellulosic CrI and cellulose DP, and enhanced the branching ratio of hemicellulose which is trapped in the cellulose. Given the distinct cell wall factors in different species, all data are grouped in standardized to eliminate the variations among plant species. The principal composition analysis and correlation analysis of the processed dataset strongly suggest that cellulose association factors determine the stem mechanical force and recalcitrance. Based on our results, we propose a model for how branches of confined hemicellulose interacted with cellulose to modulate stem strength support for the straight or bending phenotype in cut flowers.