Structural Analysis of Cell Wall Polysaccharides Using PACE.

The plant cell wall is composed of many complex polysaccharides. The composition and structure of the polysaccharides affect various cell properties including cell shape, cell function and cell adhesion. PACE (Polysaccharide Analysis using Carbohydrate gel Electrophoresis) uses a simple, rapid technique to analyze polysaccharide quantity and structure (Goubet et al.

Enrichment of the Plant Cytosolic Fraction.

The cytosol is at the core of cellular metabolism and contains many important metabolic pathways, including glycolysis, gluconeogenesis, and the pentose phosphate pathway. Despite the importance of this matrix, few attempts have sought to specifically enrich this compartment from plants. Although a variety of biochemical pathways and signaling cascades pass through the cytosol, much of the focus has usually been targeted at the reactions that occur within membrane-bound organelles of the plant cell.

Glycosylation of inositol phosphorylceramide sphingolipids is required for normal growth and reproduction in Arabidopsis.

Sphingolipids are a major component of plant plasma membranes and endomembranes, and mediate a diverse range of biological processes. Study of the highly glycosylated glycosyl inositol phosphorylceramide (GIPC) sphingolipids has been slow as a result of challenges associated with the extractability of GIPCs, and their functions in the plant remain poorly characterized. We recently discovered an Arabidopsis GIPC glucuronosyltransferase, INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE 1 (IPUT1), which is the first enzyme in the GIPC glycosylation pathway.

Glycan complexity dictates microbial resource allocation in the large intestine.

The structure of the human gut microbiota is controlled primarily through the degradation of complex dietary carbohydrates, but the extent to which carbohydrate breakdown products are shared between members of the microbiota is unclear. We show here, using xylan as a model, that sharing the breakdown products of complex carbohydrates by key members of the microbiota, such as Bacteroides ovatus, is dependent on the complexity of the target glycan. Characterization of the extensive xylan degrading apparatus expressed by B.

Probing the molecular architecture of Arabidopsis thaliana secondary cell walls using two- and three-dimensional (13)C solid state nuclear magnetic resonance spectroscopy.

The plant secondary cell wall is a thickened polysaccharide and phenolic structure, providing mechanical strength to cells, particularly in woody tissues. It is the main feedstock for the developing bioenergy and green chemistry industries. Despite the role that molecular architecture (the arrangement of biopolymers relative to each other, and their conformations) plays in dictating biomass properties, such as recalcitrance to breakdown, it is poorly understood.

The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis.

Plant cells are surrounded by a cell wall that plays a key role in plant growth, structural integrity, and defense. The cell wall is a complex and diverse structure that is mainly composed of polysaccharides. The majority of noncellulosic cell wall polysaccharides are produced in the Golgi apparatus from nucleotide sugars that are predominantly synthesized in the cytosol.

Evidence that GH115 α-glucuronidase activity, which is required to degrade plant biomass, is dependent on conformational flexibility.

The microbial degradation of the plant cell wall is an important biological process that is highly relevant to environmentally significant industries such as the bioenergy and biorefining sectors. A major component of the wall is glucuronoxylan, a β1,4-linked xylose polysaccharide that is decorated with α-linked glucuronic and/or methylglucuronic acid (GlcA/MeGlcA). Recently three members of a glycoside hydrolase family, GH115, were shown to hydrolyze MeGlcA side chains from the internal regions of xylan, an activity that has not previously been described.

Annexin 1 regulates the H2O2-induced calcium signature in Arabidopsis thaliana roots.

Hydrogen peroxide is the most stable of the reactive oxygen species (ROS) and is a regulator of development, immunity and adaptation to stress. It frequently acts by elevating cytosolic free Ca(2+) ([Ca(2+) ]cyt ) as a second messenger, with activation of plasma membrane Ca(2+) -permeable influx channels as a fundamental part of this process. At the genetic level, to date only the Ca(2) (+) -permeable Stelar K(+) Outward Rectifier (SKOR) channel has been identified as being responsive to hydrogen peroxide.

Lignin biosynthesis perturbations affect secondary cell wall composition and saccharification yield in Arabidopsis thaliana.

Background: Second-generation biofuels are generally produced from the polysaccharides in the lignocellulosic plant biomass, mainly cellulose. However, because cellulose is embedded in a matrix of other polysaccharides and lignin, its hydrolysis into the fermentable glucose is hampered. The senesced inflorescence stems of a set of 20 Arabidopsis thaliana mutants in 10 different genes of the lignin biosynthetic pathway were analyzed for cell wall composition and saccharification yield.

GUX1 and GUX2 glucuronyltransferases decorate distinct domains of glucuronoxylan with different substitution patterns.

Xylan comprises up to one-third of plant cell walls, and it influences the properties and processing of biomass. Glucuronoxylan in Arabidopsis is characterized by a linear β-(1,4)-linked backbone of xylosyl residues substituted by glucuronic acid and 4-O-methylglucuronic acid (collectively termed [Me]GlcA). The role of these substitutions remains unclear.

Identification of an additional protein involved in mannan biosynthesis.

Galactomannans comprise a β-1,4-mannan backbone substituted with α-1,6-galactosyl residues. Genes encoding the enzymes that are primarily responsible for backbone synthesis and side-chain addition of galactomannans were previously identified and characterized. To identify additional genes involved in galactomannan biosynthesis, we previously performed deep EST profiling of fenugreek (Trigonella foenum-graecum L.

Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca²+- and K+-permeable conductance in root cells.

Plant cell growth and stress signaling require Ca²⁺ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH•. In root cells, extracellular OH• activates a plasma membrane Ca²⁺-permeable conductance that permits Ca²⁺ influx.

Glycosyl transferases in family 61 mediate arabinofuranosyl transfer onto xylan in grasses.

Xylan, a hemicellulosic component of the plant cell wall, is one of the most abundant polysaccharides in nature. In contrast to dicots, xylan in grasses is extensively modified by α-(1,2)- and α-(1,3)-linked arabinofuranose. Despite the importance of grass arabinoxylan in human and animal nutrition and for bioenergy, the enzymes adding the arabinosyl substitutions are unknown.

Absence of branches from xylan in Arabidopsis gux mutants reveals potential for simplification of lignocellulosic biomass.

As one of the most abundant polysaccharides on Earth, xylan will provide more than a third of the sugars for lignocellulosic biofuel production when using grass or hardwood feedstocks. Xylan is characterized by a linear β(1,4)-linked backbone of xylosyl residues substituted by glucuronic acid, 4-O-methylglucuronic acid or arabinose, depending on plant species and cell types. The biological role of these decorations is unclear, but they have a major influence on the properties of the polysaccharide.

VASCULAR-RELATED NAC-DOMAIN6 and VASCULAR-RELATED NAC-DOMAIN7 effectively induce transdifferentiation into xylem vessel elements under control of an induction system.

We previously showed that the VASCULAR-RELATED NAC-DOMAIN6 (VND6) and VND7 genes, which encode NAM/ATAF/CUC domain protein transcription factors, act as key regulators of xylem vessel differentiation. Here, we report a glucocorticoid-mediated posttranslational induction system of VND6 and VND7. In this system, VND6 or VND7 is expressed as a fused protein with the activation domain of the herpes virus VP16 protein and hormone-binding domain of the animal glucocorticoid receptor, and the protein's activity is induced by treatment with dexamethasone (DEX), a glucocorticoid derivative.

Heme-independent soluble and membrane-associated peroxidase activity of a Zea mays annexin preparation.

Annexins are cytosolic proteins capable of reversible, Ca(2+)-dependent membrane binding or insertion. Animal annexins form and regulate Ca(2+)-permeable ion channels and may therefore participate in signaling. Zea mays (maize) annexins (ZmANN33 and ZmANN35) have recently been shown to form a Ca(2+)-permeable conductance in planar lipid bilayers and also exhibit in vitro peroxidase activity.

Voltage, reactive oxygen species and the influx of calcium.

The apical plasma membrane of young Arabidopsis root hairs has recently been found to contain a depolarisation-activated Ca(2+) channel, in addition to one activated by hyperpolarisation. The depolarisation-activated Ca(2+) channel may function in signalling but the possibility that the root hair apical plasma membrane voltage may oscillate between a hyperpolarized and depolarized state suggests a role in growth control. Plant NADPH oxidase activity has yet to be considered in models of oscillatory voltage or ionic flux despite its predicted electrogenicity and voltage dependence.

Cell wall glucomannan in Arabidopsis is synthesised by CSLA glycosyltransferases, and influences the progression of embryogenesis.

Mannans are hemicellulosic polysaccharides that have previously been implicated as structural constituents of cell walls and as storage reserves but which may serve other functions during plant growth and development. Several members of the Arabidopsis cellulose synthase-like A (CSLA) family have previously been shown to synthesise mannan polysaccharides in vitro when heterologously expressed. It has also been found that CSLA7 is essential for embryogenesis, suggesting a role for the CSLA7 product in development.

Zea mays annexins modulate cytosolic free Ca2+ and generate a Ca2+-permeable conductance.

Regulation of reactive oxygen species and cytosolic free calcium ([Ca(2+)](cyt)) is central to plant function. Annexins are small proteins capable of Ca(2+)-dependent membrane binding or membrane insertion. They possess structural motifs that could support both peroxidase activity and calcium transport.

Plant extracellular ATP signalling by plasma membrane NADPH oxidase and Ca2+ channels.

Extracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca2+ can be elevated by extracellular ATP, the mechanism is unknown.

NADPH oxidase involvement in cellular integrity.

NADPH oxidase activity is involved in plant adaptation and development. The reactive oxygen species sourced by NADPH oxidase activity may contribute to wall strength and protoplast volume adjustment. Root hair bulge apices of the NADPH oxidase mutant rhd2/Atrbohc were more robust than the kjk cellulose synthase mutant, but burst more readily than the wild type (WT).

Annexins: multifunctional components of growth and adaptation.

Plant annexins are ubiquitous, soluble proteins capable of Ca(2+)-dependent and Ca(2+)-independent binding to endomembranes and the plasma membrane. Some members of this multigene family are capable of binding to F-actin, hydrolysing ATP and GTP, acting as peroxidases or cation channels. These multifunctional proteins are distributed throughout the plant and throughout the life cycle.