Deciphering the Molecular Functions of Sterols in Cellulose Biosynthesis

Sterols play vital roles in plant growth and development, as components of membranes and as precursors to steroid hormones. Analysis of Arabidopsis mutants indicates that sterol composition is crucial for cellulose biosynthesis. Sterols are widespread in the plasma membrane (PM), suggesting a possible link between sterols and the multimeric cellulose synthase complex. In one possible scenario, molecular interactions in sterol-rich PM microdomains or another form of sterol-dependent membrane scaffolding may be critical for maintaining the correct subcellular localization, structural integrity and/or activity of the cellulose synthase machinery. Another possible link may be through steryl glucosides, which could act as primers for the attachment of glucose monomers during the synthesis of β−(1 → 4) glucan chains that form the cellulose microfibrils. This mini-review examines genetic and biochemical data supporting the link between sterols and cellulose biosynthesis in cell wall formation and explores potential approaches to elucidate the mechanism of this association

Proteomic Analysis of Plasmodesmata From Populus Cell Suspension Cultures in Relation With Callose Biosynthesis

Plasmodesmata are channels that link adjacent cells in plant tissues through which molecular exchanges take place. They are involved in multiple processes vital to plant cells, such as responses to hormonal signaling or environmental challenges including osmotic stress, wounding and pathogen attack. Despite the importance of plasmodesmata, their proteome is not well-defined. Here, we have isolated fractions enriched in plasmodesmata from cell suspension cultures of Populus trichocarpa and identified 201 proteins that are enriched in these fractions, thereby providing further insight on the multiple functions of plasmodesmata. Proteomics analysis revealed an enrichment of proteins specifically involved in responses to stress, transport, metabolism and signal transduction. Consistent with the role of callose deposition and turnover in the closure and aperture of the plasmodesmata and our proteomic analysis, we demonstrate the enrichment of callose synthase activity in the plasmodesmata represented by several gene products. A new form of calcium-independent callose synthase activity was detected, in addition to the typical calcium-dependent enzyme activity, suggesting a role of calcium in the regulation of plasmodesmata through two forms of callose synthase activities. Our report provides the first proteomic investigation of the plasmodesmata from a tree species and the direct biochemical evidence for the occurrence of several forms of active callose synthases in these structures. Data are available via ProteomeXchange with identifier PXD010692

Analyses of Extracellular Carbohydrates in Oomycetes Unveil the Existence of Three Different Cell Wall Types

[EN] Some of the most devastating plant and animal pathogens belong to the oomycete class. The cell walls of these microorganisms represent an excellent target for disease control, but their carbohydrate composition is elusive. We have undertaken a detailed cell wall analysis in 10 species from 2 major oomycete orders, the Peronosporales and the Saprolegniales, thereby unveiling the existence of 3 clearly different cell wall types: type I is devoid of N-acetylglucosamine (GlcNAc) but contains glucuronic acid and mannose; type II contains up to 5% GlcNAc and residues indicative of cross-links between cellulose and 1,3- -glucans; type III is characterized by the highest GlcNAc content (>5%) and the occurrence of unusual carbohydrates that consist of 1,6-linked GlcNAc residues. These 3 cell wall types are also distinguishable by their cellulose content and the fine structure of their 1,3- - glucans. We propose a cell wall paradigm for oomycetes that can serve as a basis for the establishment of cell wall architectural models and the further identification of cell wall subtypes. This paradigm is complementary to morphological and molecular criteria for taxonomic grouping and provides useful information for unraveling poorly understood cell wall carbohydrate biosynthetic pathways through the identification and characterization of the corresponding enzymes.SIThis work was supported by grants to V.B. from the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) (2009-515 and 2010-1807), to V.B. and J.D.-U. from the Eu- ropean Union (ITN-SAPRO-238550), and to J.D.-U. from the Spanish Ministry of Science and Innovation (CGL2009-10032)

Proteomic Analysis Identifies Markers of Exposure to cadmium Sulphide Quantum Dots (CdS QDs)

The use of cadmium sulphide quantum dot (CdS QD)-enabled products has become increasingly widespread. The prospect of their release in the environment is raising concerns. Here we have used the yeast model Saccharomyces cerevisiae to determine the potential impact of CdS QD nanoparticles on living organisms. Proteomic analyses and cell viability assays performed after 9 h exposure revealed expression of proteins involved in oxidative stress and reduced lethality, respectively, whereas oxidative stress declined, and lethality increased after 24 h incubation in the presence of CdS QDs. Quantitative proteomics using the iTRAQ approach (isobaric tags for relative and absolute quantitation) revealed that key proteins involved in essential biological pathways were differentially regulated over the time course of the experiment. At 9 h, most of the glycolytic functions increased, and the abundance of the number of heat shock proteins increased. This contrasts with the situation at 24 h where glycolytic functions, some heat shock proteins as well as oxidative phosphorylation and ATP synthesis were down-regulated. It can be concluded from our data that cell exposure to CdS QDs provokes a metabolic shift from respiration to fermentation, comparable to the situation reported in some cancer cell

BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis

[EN] Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional subunits to bridge the periplasm and the outer membrane. In bacteria, cellulose synthesis and translocation is catalyzed by the inner membrane-associated bacterial cellulose synthase (Bcs)A and BcsB subunits. Similar to alginate and poly-β-1,6 N-acetylglucosamine, bacterial cellulose is implicated in the formation of sessile bacterial communities, termed biofilms, and its synthesis is likewise stimulated by cyclic-di-GMP. Biochemical studies of exopolysaccharide synthesis are hampered by difficulties in purifying and reconstituting functional enzymes. We demonstrate robust in vitro cellulose synthesis reconstituted from purified BcsA and BcsB proteins from Rhodobacter sphaeroides. Although BcsA is the catalytically active subunit, the membrane-anchored BcsB subunit is essential for catalysis. The purified BcsA-B complex produces cellulose chains of a degree of polymerization in the range 200–300. Catalytic activity critically depends on the presence of the allosteric activator cyclicdi- GMP, but is independent of lipid-linked reactants. Our data reveal feedback inhibition of cellulose synthase by UDP but not by the accumulating cellulose polymer and highlight the strict substrate specificity of cellulose synthase for UDP-glucose. A truncation analysis of BcsB localizes the region required for activity of BcsA within its C-terminal membrane-associated domain. The reconstituted reaction provides a foundation for the synthesis of biofilm exopolysaccharides, as well as its activation by cyclic-di-GMP.SIThis work was primarily supported by The Center for LignoCellulose Structure and Formation, Energy Frontier Research Center, US Department of Energy, Office of Science, Grant DE-SC0001090 and in part (BcsB truncation studies) by NIH Grant 1R01GM101001 (awarded to J.Z.). V.B. and H.M. were supported by the Royal Institute of Technology (KTH) Advanced Carbohydrate Materials Consortium (CarboMat) funded by the Swedish Research Council Formas

The β‐1,3‐glucanosyltransferases (Gels) affect the structure of the rice blast fungal cell wall during appressorium‐mediated plant infection

[EN] The fungal wall is pivotal for cell shape and function, and in interfacial protection during host infection and environmental challenge. Here, we provide the first description of the carbohydrate composition and structure of the cell wall of the rice blast fungus Magnaporthe oryzae. We focus on the family of glucan elongation proteins (Gels) and characterize five putative β-1,3-glucan glucanosyltransferases that each carry the Glycoside Hydrolase 72 signature. We generated targeted deletion mutants of all Gel isoforms, that is, the GH72+, which carry a putative carbohydrate-binding module, and the GH72− Gels, without this motif. We reveal that M. oryzae GH72+ GELs are expressed in spores and during both infective and vegetative growth, but each individual Gel enzymes are dispensable for pathogenicity. Further, we demonstrated that a Δgel1Δgel3Δgel4 null mutant has a modified cell wall in which 1,3-glucans have a higher degree of polymerization and are less branched than the wild-type strain. The mutant showed significant differences in global patterns of gene expression, a hyper-branching phenotype and no sporulation, and thus was unable to cause rice blast lesions (except via wounded tissues). We conclude that Gel proteins play significant roles in structural modification of the fungal cell wall during appressorium-mediated plant infection.SIWe acknowledge BBSRC grant BB/J008923/1

Sequential fractionation of feruloylated hemicelluloses and oligosaccharides from wheat bran using subcritical water and xylanolytic enzymes

Wheat bran is a major by-product of cereal production that still has limited use for advanced nutritional and material applications. A sequential process using subcritical water, membrane filtration and selective enzymatic treatments has been designed for the combined fractionation of functional high molar mass hemicelluloses (over 105 g mol−1) and oligosaccharides from wheat bran. This process not only offers increased total solid yield compared with conventional protocols based on alkaline extraction, but it also preserves the inherent functionalities of the phenolic groups that substitute the carbohydrate structures of the extracted hemicelluloses. Feruloylated arabinoxylans (F-AX) with high molar mass and significant radical scavenging activity can be isolated from the subcritical water extract. Structurally different oligosaccharides, including mixed-linkage β-D-glucan oligosaccharides (BGOs) and arabinoxylo-oligosaccharides (AXOs) can be recovered from the eluent after membrane filtration. The crosslinked residue after subcritical water extraction was further treated with xylanolytic enzymes to release valuable feruloylated arabinoxylo-oligosaccharides (FAXOs). The oligo- and polysaccharide fractions isolated from this sequential process show great potential for use as prebiotic or platform chemicals, and as polymeric matrices for carbohydrate-based materials with radical scavenging properties, respectively