Insights into the cell-wall dynamics in grapevine berries during ripening and in response to biotic and abiotic stresses.

The cell wall (CW) is the dynamic structure of a plant cell, acting as a barrier against biotic and abiotic stresses. In grape berries, the modifications of pulp and skin CW during softening ensure flexibility during cell expansion and determine the final berry texture. In addition, the CW of grape berry skin is of fundamental importance for winemaking, controlling secondary metabolite extractability. Grapevine varieties with contrasting CW characteristics generally respond differently to biotic and abiotic stresses. In the context of climate change, it is important to investigate the CW dynamics occurring upon different stresses, to define new adaptation strategies. This review summarizes the molecular mechanisms underlying CW modifications during grapevine berry fruit ripening, plant-pathogen interaction, or in response to environmental stresses, also considering the most recently published transcriptomic data. Furthermore, perspectives of new biotechnological approaches aiming at modifying the CW properties based on other crops' examples are also presented.

Synergistic action of synthetic peptides and amphotericin B causes disruption of the plasma membrane and cell wall in Candida albicans.

The objective of this work was to evaluate the combination of synthetic peptides based on the γ-core motif of defensin PvD1 with amphotericin B (AmB) at different concentrations against Candida albicans. We applied the checkerboard assay using different concentrations of the commercial drug AmB and the synthetic peptides γ31-45PvD1++ and γ33-41PvD1++ against C. albicans, aiming to find combinations with synergistic interactions. Between these two interactions involving γ31-45PvD1++ and AmB, an additive effect was observed. One such interaction occurred at concentrations of 0.009 µM of peptide γ31-45PvD1++ and 13.23 µM of AmB and another condition of 0.019 µM of peptide γ31-45PvD1++ and 6.61 µM of AmB. The other two concentrations of the interaction showed a synergistic effect in the combination of synthetic peptide γ31-45PvD1++ and AmB, where the concentrations were 1.40 µM peptide γ31-45PvD1++ and 0.004 µM AmB and 0.70 µM γ31-45PvD1++ peptide and 0.002 µM AmB. We proceeded with analysis of the mechanism of action involving synergistic effects. This examination unveiled a range of impactful outcomes, including the impairment of mitochondrial functionality, compromise of cell wall integrity, DNA degradation, and a consequential decline in cell viability. We also observed that both synergistic combinations were capable of causing damage to the plasma membrane and cell wall, causing leakage of intracellular components. This discovery demonstrates for the first time that the synergistic combinations found between the synthetic peptide γ31-45PvD1++ and AmB have an antifungal effect against C. albicans, acting on the integrity of the plasma membrane and cell wall.

Freezing pre-treatment improves radio frequency explosion puffing (RFEP) quality by altering the cellular structure of purple sweet potato [Ipomoea batatas (L) Lam.].

Radio frequency explosion puffing (RFEP) is a novel oil-free puffing technique used to produce crispy textured and nutritious puffed snacks. This study aimed to investigate the effects of freezing at different temperatures (-20 °C, -40 °C, -80 °C) for14 h and freezing times (1 and 2 times) on the cellular structure of purple sweet potato and the quality of RFEP chips. The analysis of cell microstructure, conductivity, and rheology revealed that higher freezing temperatures and more freezing times resulted in increased damage to the cellular structure, leading to greater cell membrane permeability and decreased cell wall stiffness. However, excessive damage to cellular structure caused tissue structure to collapse. Compared with the control group (4 °C), the RFEP sample pre-frozen once at -40 °C had a 47.13 % increase in puffing ratio and a 61.93 % increase in crispness, while hardness decreased by 23.44 % (p < 0.05). There was no significant change in anthocyanin retention or color difference. X-ray microtomography demonstrated that the RFEP sample pre-frozen once at -40 °C exhibited a more homogeneous morphology and uniform pore distribution, resulting in the highest overall acceptability. In conclusion, freezing pre-treatment before RFEP can significantly enhance the puffing quality, making this an effective method for preparing oil-free puffing products for fruits and vegetables.

Characterization of Crude and Processed Pulp Cell Walls of Three Selected Mamey Accessions (Mammea americana L.).

Mamey (Mammea americana L.) is a tropical fleshy fruit native from the West Indies and northern South America. It is very appreciated for its flavor and color but has been little described. The present study investigates the composition and histochemistry of the pulp cell walls of three mamey accessions readily available in Martinique. The impact of pulp processing into puree on cell wall composition is evaluated. The histology and rheology of mamey puree are assessed considering these characterizations. Mamey pulp cell wall composition is dominated by highly methyl-esterified pectins (DM: 66.2-76.7%) of high molecular weight, and show few hemicelluloses, mainly xyloglucans. Processing reduced methyl-esterified uronic acid contents and gave purees with significantly different viscosities. Mamey puree was composed of polydisperse particles (20-2343 µm), which size distributions were different depending on the accession: Ti Jacques was dominated by smaller particles (50% had approximated diameters lower than 160 µm), Sonson's by larger particles (50% had approximated diameters higher than 900 µm), and Galion's had an intermediate profile. This new knowledge on mamey pulp is valuable for future works on mamey processing into new food products, even more so for those including cell wall polysaccharide-degrading enzymes.

Renewable Energy Potential: Second-Generation Biomass as Feedstock for Bioethanol Production.

Biofuels are clean and renewable energy resources gaining increased attention as a potential replacement for non-renewable petroleum-based fuels. They are derived from biomass that could either be animal-based or belong to any of the three generations of plant biomass (agricultural crops, lignocellulosic materials, or algae). Over 130 studies including experimental research, case studies, literature reviews, and website publications related to bioethanol production were evaluated; different methods and techniques have been tested by scientists and researchers in this field, and the most optimal conditions have been adopted for the generation of biofuels from biomass. This has ultimately led to a subsequent scale-up of procedures and the establishment of pilot, demo, and large-scale plants/biorefineries in some regions of the world. Nevertheless, there are still challenges associated with the production of bioethanol from lignocellulosic biomass, such as recalcitrance of the cell wall, multiple pretreatment steps, prolonged hydrolysis time, degradation product formation, cost, etc., which have impeded the implementation of its large-scale production, which needs to be addressed. This review gives an overview of biomass and bioenergy, the structure and composition of lignocellulosic biomass, biofuel classification, bioethanol as an energy source, bioethanol production processes, different pretreatment and hydrolysis techniques, inhibitory product formation, fermentation strategies/process, the microorganisms used for fermentation, distillation, legislation in support of advanced biofuel, and industrial projects on advanced bioethanol. The ultimate objective is still to find the best conditions and technology possible to sustainably and inexpensively produce a high bioethanol yield.

NKS1/ELMO4 is an integral protein of a pectin synthesis protein complex and maintains Golgi morphology and cell adhesion in Arabidopsis.

Adjacent plant cells are connected by specialized cell wall regions, called middle lamellae, which influence critical agricultural characteristics, including fruit ripening and organ abscission. Middle lamellae are enriched in pectin polysaccharides, specifically homogalacturonan (HG). Here, we identify a plant-specific Arabidopsis DUF1068 protein, called NKS1/ELMO4, that is required for middle lamellae integrity and cell adhesion. NKS1 localizes to the Golgi apparatus and loss of NKS1 results in changes to Golgi structure and function. The nks1 mutants also display HG deficient phenotypes, including reduced seedling growth, changes to cell wall composition, and tissue integrity defects. These phenotypes are comparable to qua1 and qua2 mutants, which are defective in HG biosynthesis. Notably, genetic interactions indicate that NKS1 and the QUAs work in a common pathway. Protein interaction analyses and modeling corroborate that they work together in a stable protein complex with other pectin-related proteins. We propose that NKS1 is an integral part of a large pectin synthesis protein complex and that proper function of this complex is important to support Golgi structure and function.

Construction and characterization of a hypervesiculation strain of Escherichia coli Nissle 1917.

Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and deliver microbial molecules to distant target cells in a host. OMVs secreted by probiotic probiotic strain Escherichia coli Nissle 1917 (EcN) have been reported to induce an immune response. In this study, we aimed to increase the OMV production of EcN. The double gene knockout of mlaE and nlpI was conducted in EcN because the ΔmlaEΔnlpI of experimental strain E. coli K12 showed the highest OMV production in our previous report. The ΔmlaEΔnlpI of EcN showed approximately 8 times higher OMV production compared with the parental (wild-type) strain. Quick-freeze, deep-etch replica electron microscopy revealed that plasmolysis occurred in the elongated ΔmlaEΔnlpI cells and the peptidoglycan (PG) had numerous holes. While these phenomena are similar to the findings for the ΔmlaEΔnlpI of K12, there were more PG holes in the ΔmlaEΔnlpI of EcN than the K12 strain, which were observed not only at the tip of the long axis but also in the whole PG structure. Further analysis clarified that the viability of ΔmlaEΔnlpI of EcN decreased compared with that of the wild-type. Although the amount of PG in ΔmlaEΔnlpI cells was about half of that in wild-type, the components of amino acids in PG did not change in ΔmlaEΔnlpI. Although the viability decreased compared to the wild-type, the ΔmlaEΔnlpI grew in normal culture conditions. The hypervesiculation strain constructed here is expected to be used as an enhanced probiotic strain.

A rice GT61 glycosyltransferase possesses dual activities mediating 2-O-xylosyl and 2-O-arabinosyl substitutions of xylan.

MAIN CONCLUSION: A member of the rice GT61 clade B is capable of transferring both 2-O-xylosyl and 2-O-arabinosyl residues onto xylan and another member specifically catalyses addition of 2-O-xylosyl residue onto xylan. Grass xylan is substituted predominantly with 3-O-arabinofuranose (Araf) as well as with some minor side chains, such as 2-O-Araf and 2-O-(methyl)glucuronic acid [(Me)GlcA]. 3-O-Arabinosylation of grass xylan has been shown to be catalysed by grass-expanded clade A members of the glycosyltransferase family 61. However, glycosyltransferases mediating 2-O-arabinosylation of grass xylan remain elusive. Here, we performed biochemical studies of two rice GT61 clade B members and found that one of them was capable of transferring both xylosyl (Xyl) and Araf residues from UDP-Xyl and UDP-Araf, respectively, onto xylooligomer acceptors, whereas the other specifically catalysed Xyl transfer onto xylooligomers, indicating that the former is a xylan xylosyl/arabinosyl transferase (named OsXXAT1 herein) and the latter is a xylan xylosyltransferase (named OsXYXT2). Structural analysis of the OsXXAT1- and OsXYXT2-catalysed reaction products revealed that the Xyl and Araf residues were transferred onto O-2 positions of xylooligomers. Furthermore, we demonstrated that OsXXAT1 and OsXYXT2 were able to substitute acetylated xylooligomers, but only OsXXAT1 could xylosylate GlcA-substituted xylooligomers. OsXXAT1 and OsXYXT2 were predicted to adopt a GT-B fold structure and molecular docking revealed candidate amino acid residues at the predicted active site involved in binding of the nucleotide sugar donor and the xylohexaose acceptor substrates. Together, our results establish that OsXXAT1 is a xylan 2-O-xylosyl/2-O-arabinosyl transferase and OsXYXT2 is a xylan 2-O-xylosyltransferase, which expands our knowledge of roles of the GT61 family in grass xylan synthesis.

Advances and prospects of analytic methods for bacterial transglycosylation and inhibitor discovery.

The cell wall is essential for bacteria to maintain structural rigidity and withstand external osmotic pressure. In bacteria, the cell wall is composed of peptidoglycan. Lipid II is the basic unit for constructing highly cross-linked peptidoglycan scaffolds. Transglycosylase (TGase) is the initiating enzyme in peptidoglycan synthesis that catalyzes the ligation of lipid II moieties into repeating GlcNAc-MurNAc polysaccharides, followed by transpeptidation to generate cross-linked structures. In addition to the transglycosylases in the class-A penicillin-binding proteins (aPBPs), SEDS (shape, elongation, division and sporulation) proteins are also present in most bacteria and play vital roles in cell wall renewal, elongation, and division. In this review, we focus on the latest analytical methods including the use of radioactive labeling, gel electrophoresis, mass spectrometry, fluorescence labeling, probing undecaprenyl pyrophosphate, fluorescence anisotropy, ligand-binding-induced tryptophan fluorescence quenching, and surface plasmon resonance to evaluate TGase activity in cell wall formation. This review also covers the discovery of TGase inhibitors as potential antibacterial agents. We hope that this review will give readers a better understanding of the chemistry and basic research for the development of novel antibiotics.

Poacic Acid, a Plant-Derived Stilbenoid, Augments Cell Wall Chitin Production, but Its Antifungal Activity Is Hindered by This Polysaccharide and by Fungal Essential Metals.

Climate and environmental changes have modified the habitats of fungal pathogens, inflicting devastating effects on livestock and crop production. Additionally, drug-resistant fungi are increasing worldwide, driving the urgent need to identify new molecular scaffolds for the development of antifungal agents for humans, animals, and plants. Poacic acid (PA), a plant-derived stilbenoid, was recently discovered to be a novel molecular scaffold that inhibits the growth of several fungi. Its antifungal activity has been associated with perturbation of the production/assembly of the fungal cell wall ß-1,3-glucan, but its mode of action is not resolved. In this study, we investigated the antifungal activity of PA and its derivatives on a panel of yeast. PA had a fungistatic effect on S. cerevisiae and a fungicidal effect on plasma membrane-damaged Candida albicans mutants. Live cell fluorescence microscopy experiments revealed that PA increases chitin production and modifies its cell wall distribution. Chitin production and cell growth returned to normal after prolonged incubation. The antifungal activity of PA was reduced in the presence of exogenous chitin, suggesting that the potentiation of chitin production is a stress response that helps the yeast cell overcome the effect of this antifungal stilbenoid. Growth inhibition was also reduced by metal ions, indicating that PA affects the metal homeostasis. These findings suggest that PA has a complex antifungal mechanism of action that involves perturbation of the cell wall ß-1,3-glucan production/assembly, chitin production, and metal homeostasis.

Structure of the Bacterial Cell Envelope and Interactions with Antimicrobials: Insights from Molecular Dynamics Simulations.

Bacteria have evolved over 3 billion years, shaping our intrinsic and symbiotic coexistence with these single-celled organisms. With rising populations of drug-resistant strains, the search for novel antimicrobials is an ongoing area of research. Advances in high-performance computing platforms have led to a variety of molecular dynamics simulation strategies to study the interactions of antimicrobial molecules with different compartments of the bacterial cell envelope of both Gram-positive and Gram-negative species. In this review, we begin with a detailed description of the structural aspects of the bacterial cell envelope. Simulations concerned with the transport and associated free energy of small molecules and ions through the outer membrane, peptidoglycan, inner membrane and outer membrane porins are discussed. Since surfactants are widely used as antimicrobials, a section is devoted to the interactions of surfactants with the cell wall and inner membranes. The review ends with a discussion on antimicrobial peptides and the insights gained from the molecular simulations on the free energy of translocation. Challenges involved in developing accurate molecular models and coarse-grained strategies that provide a trade-off between atomic details with a gain in sampling time are highlighted. The need for efficient sampling strategies to obtain accurate free energies of translocation is also discussed. Molecular dynamics simulations have evolved as a powerful tool that can potentially be used to design and develop novel antimicrobials and strategies to effectively treat bacterial infections.

Lipoarabinomannan mediates localized cell wall integrity during division in mycobacteria.

The growth and division of mycobacteria, which include clinically relevant pathogens, deviate from that of canonical bacterial models. Despite their Gram-positive ancestry, mycobacteria synthesize and elongate a diderm envelope asymmetrically from the poles, with the old pole elongating more robustly than the new pole. The phosphatidylinositol-anchored lipoglycans lipomannan (LM) and lipoarabinomannan (LAM) are cell envelope components critical for host-pathogen interactions, but their physiological functions in mycobacteria remained elusive. In this work, using biosynthetic mutants of these lipoglycans, we examine their roles in maintaining cell envelope integrity in Mycobacterium smegmatis and Mycobacterium tuberculosis. We find that mutants defective in producing mature LAM fail to maintain rod cell shape specifically at the new pole and para-septal regions whereas a mutant that produces a larger LAM becomes multi-septated. Therefore, LAM plays critical and distinct roles at subcellular locations associated with division in mycobacteria, including maintenance of local cell wall integrity and septal placement.

Characterization of the fiber-like cortical cells in moss gametophytes.

MAIN CONCLUSION: Fiber-like cells with thickened cell walls of specific structure and polymer composition that includes (1 → 4)-ß-galactans develop in the outer stem cortex of several moss species gametophytes. The early land plants evolved several specialized cell types and tissues that did not exist in their aquatic ancestors. Of these, water-conducting elements and reproductive organs have received most of the research attention. The evolution of tissues specialized to fulfill a mechanical function is by far less studied despite their wide distribution in land plants. For vascular plants following a homoiohydric trajectory, the evolutionary emergence of mechanical tissues is mainly discussed starting with the fern-like plants with their hypodermal sterome or sclerified fibers that have xylan and lignin-based cell walls. However, mechanical challenges were also faced by bryophytes, which lack lignified cell-walls. To characterize mechanical tissues in the bryophyte lineage, following a poikilohydric trajectory, we used six wild moss species (Polytrichum juniperinum, Dicranum sp., Rhodobryum roseum, Eurhynchiadelphus sp., Climacium dendroides, and Hylocomium splendens) and analyzed the structure and composition of their cell walls. In all of them, the outer stem cortex of the leafy gametophytic generation had fiber-like cells with a thickened but non-lignified cell wall. Such cells have a spindle-like shape with pointed tips. The additional thick cell wall layer in those fiber-like cells is composed of sublayers with structural evidence for different cellulose microfibril orientation, and with specific polymer composition that includes (1 → 4)-ß-galactans. Thus, the basic cellular characters of the cells that provide mechanical support in vascular plant taxa (elongated cell shape, location at the periphery of a primary organ, the thickened cell wall and its peculiar composition and structure) also exist in mosses.

Monitoring the Interaction of the Peptidoglycan with the Bacterial ß-Barrel Assembly Machinery.

Gram-negative bacteria coordinate the biosynthesis of their different cell envelope components. Growth of the outer membrane (OM) requires the essential ß-barrel assembly machine (BAM), which inserts OM proteins (OMPs) into the OM. The underlying peptidoglycan (PG) sacculus grows by the insertion of nascent glycan chains. We have previously identified interactions between BAM and PG in E. coli and showed that these interactions coordinate OM biogenesis with PG growth. BAM responds to the maturation state of the PG, and this mechanism activates preferentially BAM complexes at sites of active PG synthesis. Here we present protocols to purify soluble Bam proteins and full-length BamABCDE, isolate PG and soluble PG fragments, and study BAM-PG interactions with the isolated components. We also describe the protocol to detect interactions between Bam proteins and PG in cells.

WOX2 functions redundantly with WOX1 and WOX4 to positively regulate seed germination in Arabidopsis.

MAIN CONCLUSION: WOX family gene WOX2 is highly expressed during seed development, which functions redundantly with WOX1 and WOX4 to positively regulate seed germination. WOX (WUSCHEL-related homeobox) is a family of transcription factors in plants. They play essential roles in the regulation of plant growth and development, but their function in seed germination is not well understood. In this report, we show that WOX1, WOX2, and WOX4 are close homologues in Arabidopsis. WOX2 has a redundant function with WOX1 and WOX4, respectively, in seed germination. WOX2 is highly expressed during seed development, from the globular embryonic stage to mature dry seeds, and its expression is decreased after germination. Loss of function single mutant wox2, and double mutants wox1 wox2 and wox2 wox4-1 show decreased germination speed. WOX2 and WOX4 are essential for hypocotyl-radicle zone elongation during germination, potentially by promoting the expression of cell wall-related genes. We also found that WOX2 and WOX4 regulate germination through the gibberellin (GA) pathway. These results suggest that WOX2 and WOX4 integrate the GA pathway and downstream cell wall-related genes during germination.

Preparation of isolated guard cells, containing cell walls, from Vicia faba.

Stomatal movement, initiated by specialized epidermal cells known as guard cells (GCs), plays a pivotal role in plant gas exchange and water use efficiency. Despite protocols existing for isolating GCs through proplasting for carrying out biochemical, physiological, and molecular studies, protocals for isolating GCs with their cell walls still intact have been lacking in the literature. In this paper, we introduce a method for the isolation of complete GCs from Vicia faba and show their membrane to remain impermeable through propidium iodide staining. This methodology enables further in-depth analyses into the cell wall composition of GCs, facilitating our understanding of structure-function relationship governing reversible actuation within cells.

Vegetative cell wall protein OsGP1 regulates cell wall mediated soda saline-alkali stress in rice.

Plant growth and development are inhibited by the high levels of ions and pH due to soda saline-alkali soil, and the cell wall serves as a crucial barrier against external stresses in plant cells. Proteins in the cell wall play important roles in plant cell growth, morphogenesis, pathogen infection and environmental response. In the current study, the full-length coding sequence of the vegetative cell wall protein gene OsGP1 was characterized from Lj11 (Oryza sativa longjing11), it contained 660 bp nucleotides encoding 219 amino acids. Protein-protein interaction network analysis revealed possible interaction between CESA1, TUBB8, and OsJ_01535 proteins, which are related to plant growth and cell wall synthesis. OsGP1 was found to be localized in the cell membrane and cell wall. Furthermore, overexpression of OsGP1 leads to increase in plant height and fresh weight, showing enhanced resistance to saline-alkali stress. The ROS (reactive oxygen species) scavengers were regulated by OsGP1 protein, peroxidase and superoxide dismutase activities were significantly higher, while malondialdehyde was lower in the overexpression line under stress. These results suggest that OsGP1 improves saline-alkali stress tolerance of rice possibly through cell wall-mediated intracellular environmental homeostasis.

Lightweight and Strong Cellulosic Triboelectric Materials Enabled by Cell Wall Nanoengineering.

As intelligent technology surges forward, wearable electronics have emerged as versatile tools for monitoring health and sensing our surroundings. Among these advancements, porous triboelectric materials have garnered significant attention for their lightness. However, these materials face the challenge of improving structural stability to further enhance the sensing accuracy of triboelectric sensors. In this study, a lightweight and strong porous cellulosic triboelectric material is designed by cell wall nanoengineering. By tailoring of the cell wall structure, the material shows a high mechanical strength of 51.8 MPa. The self-powered sensor constructed by this material has a high sensitivity of 33.61 kPa-1, a fast response time of 36 ms, and excellent pressure detection durability. Notably, the sensor still enables a high sensing performance after the porous cellulosic triboelectric material exposure to 200 °C and achieves real-time feedback of human motion, thereby demonstrating great potential in the field of wearable electronic devices.

Characterization of a Novel Antimicrobial Peptide Bacipeptin against Foodborne Pathogens.

The increasing emergence of multidrug-resistant pathogens and development of biopreservatives in food industries has increased the demand of novel and safe antimicrobial agents. In this study, a marine bacterial strain Bacillus licheniformis M1 was isolated and exhibited obvious antimicrobial activities against foodborne pathogens, especially against methicillin-resistant Staphylococcus aureus. The antimicrobial agent was purified and identified as a novel antimicrobial peptide, which was designated as bacipeptin, and the corresponding mechanism was further investigated by electron microscopy observation and transcriptomic analysis with biochemical validation. The results showed that bacipeptin could reduce the virulence of methicillin-resistant Staphylococcus aureus and exerted its antimicrobial activity by interfering with histidine metabolism, inducing the accumulation of reactive oxygen species and down-regulating genes related to Na+/H+ antiporter and the cell wall, thus causing damage to the cell wall and membrane. Overall, our study provides a novel natural product against foodborne pathogens and discloses the corresponding action mechanism.

Importance of Non-Covalent Interactions in Yeast Cell Wall Molecular Organization.

This review covers a group of non-covalently associated molecules, particularly proteins (NCAp), incorporated in the yeast cell wall (CW) with neither disulfide bridges with proteins covalently attached to polysaccharides nor other covalent bonds. Most NCAp, particularly Bgl2, are polysaccharide-remodeling enzymes. Either directly contacting their substrate or appearing as CW lipid-associated molecules, such as in vesicles, they represent the most movable enzymes and may play a central role in CW biogenesis. The absence of the covalent anchoring of NCAp allows them to be there where and when it is necessary. Another group of non-covalently attached to CW molecules are polyphosphates (polyP), the universal regulators of the activity of many enzymes. These anionic polymers are able to form complexes with metal ions and increase the diversity of non-covalent interactions through charged functional groups with both proteins and polysaccharides. The mechanism of regulation of polysaccharide-remodeling enzyme activity in the CW is unknown. We hypothesize that polyP content in the CW is regulated by another NCAp of the CW-acid phosphatase-which, along with post-translational modifications, may thus affect the activity, conformation and compartmentalization of Bgl2 and, possibly, some other polysaccharide-remodeling enzymes.