The Cell Wall Arabinose-Deficient Arabidopsis thaliana Mutant murus5 Encodes a Defective Allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2.

Traditional marker-based mapping and next-generation sequencing was used to determine that the Arabidopsis (Arabidopsis thaliana) low cell wall arabinose mutant murus5 (mur5) encodes a defective allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2 (RGP2). Marker analysis of 13 F2 confirmed mutant progeny from a recombinant mapping population gave a rough map position on the upper arm of chromosome 5, and deep sequencing of DNA from these 13 lines gave five candidate genes with G→A (C→T) transitions predicted to result in amino acid changes. Of these five, only insertional mutant alleles of RGP2, a gene that encodes a UDP-arabinose mutase that interconverts UDP-arabinopyranose and UDP-arabinofuranose, exhibited the low cell wall arabinose phenotype.

Galactose-depleted xyloglucan is dysfunctional and leads to dwarfism in Arabidopsis.

Xyloglucan is a polysaccharide that has important roles in the formation and function of the walls that surround growing land plant cells. Many of these plants synthesize xyloglucan that contains galactose in two different side chains (L and F), which exist in distinct molecular environments. However, little is known about the contribution of these side chains to xyloglucan function.

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.

Bifunctional cytosolic UDP-glucose 4-epimerases catalyse the interconversion between UDP-D-xylose and UDP-L-arabinose in plants.

UDP-sugars serve as substrates in the synthesis of cell wall polysaccharides and are themselves generated through sequential interconversion reactions from UDP-Glc (UDP-glucose) as the starting substrate in the cytosol and the Golgi apparatus. For the present study, a soluble enzyme with UDP-Xyl (UDP-xylose) 4-epimerase activity was purified approx. 300-fold from pea (Pisum sativum L.

Characterization of Arabidopsis mur3 mutations that result in constitutive activation of defence in petioles, but not leaves.

A screen was established for mutants in which the plant defence response is de-repressed. The pathogen-inducible isochorismate synthase (ICS1) promoter was fused to firefly luciferase (luc) and a homozygous transgenic line generated in which the ICS1:luc fusion is co-regulated with ICS1. This line was mutagenized and M(2) seedlings screened for constitutive ICS1:luc expression (cie).

Biochemical genetics of nucleotide sugar interconversion reactions.

During the past few years, substantial progress has been made to understand the enzymology and regulation of nucleotide sugar interconversion reactions that are irreversible in vivo on thermodynamic grounds. Feedback inhibition of enzymes by metabolic end products appears to be a common theme but some experimental results on recombinant enzymes are difficult to interpret. Using a combination of metabolic flux analysis, enzyme assays, and bioinformatics approaches, the significance of several proposed alternate pathways has been clarified.

The Arabidopsis root hair cell wall formation mutant lrx1 is suppressed by mutations in the RHM1 gene encoding a UDP-L-rhamnose synthase.

Cell and cell wall growth are mutually dependent processes that must be tightly coordinated and controlled. LRR-extensin1 (LRX1) of Arabidopsis thaliana is a potential regulator of cell wall development, consisting of an N-terminal leucine-rich repeat domain and a C-terminal extensin-like domain typical for structural cell wall proteins. LRX1 is expressed in root hairs, and lrx1 mutant plants develop distorted root hairs that often swell, branch, or collapse.

Depletion of UDP-D-apiose/UDP-D-xylose synthases results in rhamnogalacturonan-II deficiency, cell wall thickening, and cell death in higher plants.

D-apiose serves as the binding site for borate cross-linking of rhamnogalacturonan II (RG-II) in the plant cell wall, and biosynthesis of D-apiose involves UDP-D-apiose/UDP-D-xylose synthase catalyzing the conversion of UDP-D-glucuronate to a mixture of UDP-D-apiose and UDP-D-xylose. In this study we have analyzed the cellular effects of depletion of UDP-D-apiose/UDP-D-xylose synthases in plants by using virus-induced gene silencing (VIGS) of NbAXS1 in Nicotiana benthamiana. The recombinant NbAXS1 protein exhibited UDP-D-apiose/UDP-D-xylose synthase activity in vitro.

The biosynthesis of D-Galacturonate in plants. functional cloning and characterization of a membrane-anchored UDP-D-Glucuronate 4-epimerase from Arabidopsis.

Pectic cell wall polysaccharides owe their high negative charge to the presence of D-galacturonate, a monosaccharide that appears to be present only in plants and some prokaryotes. UDP-D-galacturonate, the activated form of this sugar, is known to be formed by the 4-epimerization of UDP-D-glucuronate; however, no coding regions for the epimerase catalyzing this reaction have previously been described in plants. To better understand the mechanisms by which precursors for pectin synthesis are produced, we used a bioinformatics approach to identify and functionally express a UDP-D-glucuronate 4-epimerase (GAE1) from Arabidopsis.

Identification and characterization of a UDP-D-glucuronate 4-epimerase in Arabidopsis.

One of the major sugars present in the plant cell wall is d-galacturonate, the dominant monosaccharide in pectic polysaccharides. Previous work indicated that one of the activated precursors necessary for the synthesis of pectins is UDP-d-galacturonate, which is synthesized from UDP-d-glucuronate by a UDP-d-glucuronate 4-epimerase (GAE). Here, we report the identification, cloning and characterization of a GAE6 from Arabidopsis thaliana.

Molecular analysis of 10 coding regions from Arabidopsis that are homologous to the MUR3 xyloglucan galactosyltransferase.

Plant cell walls are composed of a large number of complex polysaccharides, which contain at least 13 different monosaccharides in a multitude of linkages. This structural complexity of cell wall components is paralleled by a large number of predicted glycosyltransferases in plant genomes, which can be grouped into several distinct families based on conserved sequence motifs (B. Henrissat, G.

The biosynthesis of the branched-chain sugar d-apiose in plants: functional cloning and characterization of a UDP-d-apiose/UDP-d-xylose synthase from Arabidopsis.

d-Apiose is a plant-specific branched-chain monosaccharide found in rhamnogalacturonan II (RG-II), apiogalacturonan, and several apioglycosides. Within RG-II, d-apiose serves as the binding site for borate, which leads to the formation of cross-links within the wall. Biochemical studies in duckweed and parsley have established that uridine 5'-diphospho-d-apiose (UDP-d-apiose) is formed from UDP-d-glucuronate by decarboxylation and re-arrangement of the carbon skeleton, leading to ring contraction and branch formation.

The MUR3 gene of Arabidopsis encodes a xyloglucan galactosyltransferase that is evolutionarily related to animal exostosins.

Xyloglucans are the principal glycans that interlace cellulose microfibrils in most flowering plants. The mur3 mutant of Arabidopsis contains a severely altered structure of this polysaccharide because of the absence of a conserved alpha-L-fucosyl-(1-->2)-beta-D-galactosyl side chain and excessive galactosylation at an alternative xylose residue. Despite this severe structural alteration, mur3 plants were phenotypically normal and exhibited tensile strength in their inflorescence stems comparable to that of wild-type plants.

Tensile properties of Arabidopsis cell walls depend on both a xyloglucan cross-linked microfibrillar network and rhamnogalacturonan II-borate complexes.

The mechanical properties of plant organs depend upon anatomical structure, cell-cell adhesion, cell turgidity, and the mechanical properties of their cell walls. By testing the mechanical responses of Arabidopsis mutants, it is possible to deduce the contribution that polymers of the cell wall make to organ strength. We developed a method to measure the tensile parameters of the expanded regions of turgid or plasmolyzed dark-grown Arabidopsis hypocotyls and applied it to the fucose biosynthesis mutant mur1, the xyloglucan glycosyltransferase mutants mur2 and mur3, and the katanin mutant bot1.

The GMD1 and GMD2 genes of Arabidopsis encode isoforms of GDP-D-mannose 4,6-dehydratase with cell type-specific expression patterns.

l-Fucose (l-Fuc) is a monosaccharide constituent of plant cell wall polysaccharides and glycoproteins. The committing step in the de novo synthesis of l-Fuc is catalyzed by GDP-d-mannose 4,6-dehydratase, which, in Arabidopsis, is encoded by the GMD1 and GMD2 (MUR1) genes. To determine the functional significance of this genetic redundancy, the expression patterns of both genes were investigated via promoter-beta-glucuronidase fusions and immunolocalization of a Fuc-containing epitope.

Distribution of fucose-containing xyloglucans in cell walls of the mur1 mutant of Arabidopsis.

The monoclonal antibody, CCRC-M1, which recognizes a fucose (Fuc)-containing epitope found principally in the cell wall polysaccharide xyloglucan, was used to determine the distribution of this epitope throughout the mur1 mutant of Arabidopsis. Immunofluorescent labeling of whole seedlings revealed that mur1 root hairs are stained heavily by CCRC-M1, whereas the body of the root remains unstained or only lightly stained. Immunogold labeling showed that CCRC-M1 labeling within the mur1 root is specific to particular cell walls and cell types.

The biosynthesis of L-arabinose in plants: molecular cloning and characterization of a Golgi-localized UDP-D-xylose 4-epimerase encoded by the MUR4 gene of Arabidopsis.

The mur4 mutant of Arabidopsis shows a 50% reduction in the monosaccharide L-Ara in leaf-derived cell wall material because of a partial defect in the 4-epimerization of UDP-D-Xyl to UDP-L-Ara. To determine the genetic lesion underlying the mur4 phenotype, the MUR4 gene was cloned by a map-based procedure and found to encode a type-II membrane protein with sequence similarity to UDP-D-Glc 4-epimerases. Enzyme assays of MUR4 protein expressed in the methylotropic yeast Pichia pastoris indicate that it catalyzes the 4-epimerization of UDP-D-Xyl to UDP-L-Ara, the nucleotide sugar used by glycosyltransferases in the arabinosylation of cell wall polysaccharides and wall-resident proteoglycans.

Structure of the MUR1 GDP-mannose 4,6-dehydratase from Arabidopsis thaliana: implications for ligand binding and specificity.

GDP-D-mannose 4,6-dehydratase catalyzes the first step in the de novo synthesis of GDP-L-fucose, the activated form of L-fucose, which is a component of glycoconjugates in plants known to be important to the development and strength of stem tissues. We have determined the three-dimensional structure of the MUR1 dehydratase isoform from Arabidopsis thaliana complexed with its NADPH cofactor as well as with the ligands GDP and GDP-D-rhamnose. MUR1 is a member of the nucleoside-diphosphosugar modifying subclass of the short-chain dehydrogenase/reductase enzyme family, having homologous structures and a conserved catalytic triad of Lys, Tyr, and Ser/Thr residues.

Biosynthesis and properties of the plant cell wall.

The characterization of cell wall mutants of Arabidopsis thaliana, combined with biochemical approaches toward the purification and characterization of glycosyltransferases, has led to significant advances in understanding cell wall synthesis and the properties of cell walls. New insights have been gained into the formation of cellulose and the functions of the matrix polysaccharides rhamnogalacturonan-II and xyloglucan.

Mobilization of seed storage lipid by Arabidopsis seedlings is retarded in the presence of exogenous sugars.

Background: Soluble sugar levels must be closely regulated in germinating seeds to ensure an adequate supply of energy and building materials for the developing seedling. Studies on germinating cereal seeds indicate that production of sugars from starch is inhibited by increasing sugar levels. Although numerous studies have focused on the regulation of starch metabolism, very few studies have addressed the control of storage lipid metabolism by germinating oilseeds.

The mur2 mutant of Arabidopsis thaliana lacks fucosylated xyloglucan because of a lesion in fucosyltransferase AtFUT1.

Cell walls of the Arabidopsis mutant mur2 contain less than 2% of the wild-type amount of fucosylated xyloglucan because of a point mutation in the fucosyltransferase AtFUT1. The mur2 mutation eliminates xyloglucan fucosylation in all major plant organs, indicating that Arabidopsis thaliana fucosyltransferase 1 (AtFUT1) accounts for all of the xyloglucan fucosyltransferase activity in Arabidopsis. Despite this alteration in structure, mur2 plants show a normal growth habit and wall strength.