Showing papers by "Natasha V. Raikhel published in 2002"

CLV3 Is Localized to the Extracellular Space, Where It Activates the Arabidopsis CLAVATA Stem Cell Signaling Pathway

Abstract: Plant growth and development depends on the activity of a continuously replenished pool of stem cells within the shoot apical meristem to supply cells for organogenesis. In Arabidopsis, the stem cell–specific protein CLAVATA3 (CLV3) acts cell nonautonomously to restrict the size of the stem cell population, but the hypothesis that CLV3 acts as an extracellular signaling molecule has not been tested. We used genetic and immunological assays to show that CLV3 localizes to the apoplast and that export to the extracellular space is required for its function in activating the CLV1/CLV2 receptor complex. Apoplastic localization allows CLV3 to signal from the stem cell population to the organizing center in the underlying cells.

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

Abstract: 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. In contrast, Arabidopsis mur1 mutants that are defective in the de novo synthesis of l-fucose exhibit a dwarfed growth habit and decreased wall strength [Reiter, W. D., Chapple, C. & Somerville, C. R. (1993) Science 261, 1032–1035]. Because the mur1 mutation affects several cell wall polysaccharides, whereas the mur2 mutation is specific to xyloglucan, the phenotypes of mur1 plants appear to be caused by structural changes in fucosylated pectic components such as rhamnogalacturonan-II. The normal growth habit and wall strength of mur2 plants casts doubt on hypotheses regarding roles of xyloglucan fucosylation in facilitating xyloglucan–cellulose interactions or in modulating growth regulator activity.

An Arabidopsis gene encoding an α-xylosyltransferase involved in xyloglucan biosynthesis

Abstract: Microsomal membranes catalyze the formation of xyloglucan from UDP-Glc and UDP-Xyl by cooperative action of α-xylosyltransferase and β-glucan synthase activities. Here we report that etiolated pea microsomes contain an α-xylosyltransferase that catalyzes the transfer of xylose from UDP-[14C]xylose onto β(1,4)-linked glucan chains. The solubilized enzyme had the capacity to transfer xylosyl residues onto cello-oligosaccharides having 5 or more glucose residues. Analysis of the data from these biochemical assays led to the identification of a group of Arabidopsis genes and the hypothesis that one or more members of this group may encode α-xylosyltransferases involved in xyloglucan biosynthesis. To evaluate this hypothesis, the candidate genes were expressed in Pichia pastoris and their activities measured with the biochemical assay described above. One of the candidate genes showed cello-oligosaccharide-dependent xylosyltransferase activity. Characterization of the radiolabeled products obtained with cellopentaose as acceptor indicated that the pea and the Arabidopsis enzymes transfer the 14C-labeled xylose mainly to the second glucose residue from the nonreducing end. Enzymatic digestion of these radiolabeled products produced results that would be expected if the xylose was attached in an α(1,6)-linkage to the glucan chain. We conclude that this Arabidopsis gene encodes an α-xylosyltransferase activity involved in xyloglucan biosynthesis.

NPSN11 Is a Cell Plate-Associated SNARE Protein That Interacts with the Syntaxin KNOLLE

Abstract: SNAREs are important components of the vesicle trafficking machinery in eukaryotic cells. In plants, SNAREs have been found to play a variety of roles in the development and physiology of the whole organism. Here, we describe the identification and characterization of a novel plant-specific SNARE, NPSN11, a member of a closely related small gene family in Arabidopsis. NSPN11 is highly expressed in actively dividing cells. In a subcellular fractionation experiment, NSPN11 cofractionates with the cytokinesis-specific syntaxin, KNOLLE, which is required for the formation of the cell plate. By immunofluorescence microscopy, NSPN11 was localized to the cell plate in dividing cells. Consistent with the localization studies, NSPN11 was found to interact with KNOLLE. Our results suggest that NPSN11 is another component of the membrane trafficking and fusion machinery involved in cell plate formation.

Our New Impact Factor

Abstract: Plant Physiology's impact factor rose from 4.831 in 2000 to 5.105 in 2001—an increase of 5.67%! For those who do not know, the impact factor of a journal (for 2001) is calculated by dividing the number of citations made in 2001 to articles published in a journal in 1999 through 2000 by the items

Molecular Genetics of Non-processive Glycosyltransferases

Abstract: Although Arabidopsis has become the model for plant genomic and developmental research, it remains behind the rest of the field with respect to biochemical and structural analyses. However, the availability of the genome sequence provides an alternative and probable first approach towards identification, by homology, of potentially interesting genes. Subsequently, such a genetic approach has been useful in identifying a class of enzymes involved in cell wall biosynthesis, the polysaccharide glycosyltransferases. Most enzymes that are involved in glycan biosynthesis are glycosyltransferases. These enzymes act mainly by adding monosaccharides (donor molecules) one at a time to specific positions on acceptor molecules and thus assemble monosaccharides into linear and branched sugar chains. It has been estimated that more than 100 distinct glycosidic linkages are present in the glycoconjugate repertoire of any given multicellular organism. Because each linkage is usually the product of a different glycosyltransferase, it appears that a large number of distinct glycosyltransferases is present in higher eukaryotes. Many, but not all, of these enzymes are found within the ER-Golgi pathway where the synthesis of many glycoconjugates occurs. Glycosyltransferases may act as either processive or non-processive enzymes. Processive glycosyltransferases (often referred to as polysaccharide synthases) add sugar moieties to the growing end of a linear polysaccharide without releasing the acceptor substrate. One classic example is cellulose synthase. Non-processive glycosyltransferases add single monosaccharides to specific positions of an acceptor molecule, then move on to other acceptor sites. These enzymes are usually type II membrane proteins possessing a single hydrophobic segment spanning the Golgi membrane, which functions as a signal-anchor sequence. A short amino-terminal portion faces the cytosol and the carboxy-terminal catalytic domain is positioned within the lumen of the Golgi apparatus (Figure 1). Figure 1. Schematic diagram of most Golgi-resident glycosyltransferases. They are characterized by a short NH2-terminus oriented toward the cytoplasmic side of the Golgi membrane, a single membrane spanning region, and a variable length stem connecting the globular ... Many glycosyltransferases have been identified and extensively studied in animal, fungal, and bacterial systems; these are classified into different families, based on the type of sugar that they transfer and the type of linkage that they establish. For instance, an enzyme transferring a galactose residue from UDP-D-galactose onto the 2-hydroxyl group of another sugar moiety would be considered a (1,2)-galactosyltransferase. The sugar moiety may be added in either alpha or beta configuration adding further complexity to the types of linkages that can be formed. If in the above example the newly attached galactose residue is in the beta configuration, the enzyme would be classified as a *b(1,2)-galactosyltransferase. Since the sugar moiety in UDP-D-galactose exists in the alpha configuration, b-galactosyltransferases act with inversion of configuration whereas a-galactosyltransferases act with retention of configuration. Like other eukaryotic organisms, plants have glycoproteins and glycolipids; however, a major feature distinguishing plants from animals is the presence of a complex wall surrounding every cell. These walls play a crucial role in development, signal transduction, and response to environmental factors, including microbial pathogens and insects. Plant cell walls are mainly composed of cellulose microfibrils and matrix polysaccharides that encompass hemicelluloses and pectins. The synthesis of polysaccharides can be divided into two main stages: synthesis of the backbone, performed by polysaccharide synthases, and addition of side-chain residues mediated by glycosyltransferases. The structural complexity of many of these polysaccharides is attributed to numerous modifications that involve additions of various sugars by glycosyltransferases followed by acetylations, methylations, and addition of phenolics. Although biochemical analyses have demonstrated the existence of many glycosyltransferases in plants (White et al., 1993; Keegstra & Raikhel, 2001 and references therein), to date only three genes encoding non-processive glycosyltransferases in cell wall polysaccharide biosynthesis have been cloned, and several genes that are involved in glycoprotein biosynthesis have been identified as well. Low amino acid sequence similarity between families of glycosyltransferases and the difficulties of obtaining acceptor substrates contribute to the lack of success in identifying plant cell wall glycosyltransferases. In this chapter we will highlights some of the advances made in this area over the past few years.