title: Subcompartment localization of the side chain xyloglucan-synthesizing enzymes within Golgi stacks of tobacco suspension-cultured cells.
abstract: Xyloglucan is the dominant hemicellulosic polysaccharide of the primary cell wall of dicotyledonous plants that plays a key role in plant development. It is well established that xyloglucan is assembled within Golgi stacks and transported in Golgi-derived vesicles to the cell wall. It is also known that the biosynthesis of xyloglucan requires the action of glycosyltransferases including α-1,6-xylosyltransferase, β-1,2-galactosyltransferase and α-1,2-fucosyltransferase activities responsible for the addition of xylose, galactose and fucose residues to the side chains. There is, however, a lack of knowledge on how these enzymes are distributed within subcompartments of Golgi stacks. We have undertaken a study aiming at mapping these glycosyltransferases within Golgi stacks using immunogold-electron microscopy. To this end, we generated transgenic lines of tobacco (Nicotiana tabacum) BY-2 suspension-cultured cells expressing either the α-1,6-xylosyltransferase, AtXT1, the β-1,2-galactosyltransferase, AtMUR3, or the α-1,2-fucosyltransferase AtFUT1 of Arabidopsis thaliana fused to green-fluorescent protein (GFP). Localization of the fusion proteins within the endomembrane system was assessed using confocal microscopy. Additionally, tobacco cells were high pressure-frozen/freeze-substituted and subjected to quantitative immunogold labelling using anti-GFP antibodies to determine the localization patterns of the enzymes within subtypes of Golgi cisternae. The data demonstrate that: (i) all fusion proteins, AtXT1-GFP, AtMUR3-GFP and AtFUT1-GFP are specifically targeted to the Golgi apparatus; and (ii) AtXT1-GFP is mainly located in the cis and medial cisternae, AtMUR3-GFP is predominantly associated with medial cisternae and AtFUT1-GFP mostly detected over trans cisternae suggesting that initiation of xyloglucan side chains occurs in early Golgi compartments in tobacco cells.
authors.affiliation: Laboratoire 'Glycobiologie et Matrice Extracellulaire Végétale,' UPRES EA 4358, Institut Fédératif de Recherche Multidisciplinaire sur les Peptides 23, Plate-forme de Recherche en Imagerie Cellulaire de Haute Normandie (PRIMACEN), IBiSA, Université de Rouen, 76821 Mont-Saint Aignan Cedex, France.
authors.forename: Laurence
authors.initials: L
authors.lastname: Chevalier
authors.forename: Sophie
authors.initials: S
authors.lastname: Bernard
authors.forename: Yasmina
authors.initials: Y
authors.lastname: Ramdani
authors.forename: Romain
authors.initials: R
authors.lastname: Lamour
authors.forename: Muriel
authors.initials: M
authors.lastname: Bardor
authors.forename: Patrice
authors.initials: P
authors.lastname: Lerouge
authors.forename: Marie-Laure
authors.initials: ML
authors.lastname: Follet-Gueye
authors.forename: Azeddine
authors.initials: A
authors.lastname: Driouich
mesh_terms: D002478:Cells, Cultured; D005647:Fucosyltransferases / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism; D005936:Glucans / Q000096:biosynthesis*; D006056:Golgi Apparatus / Q000201:enzymology* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000378:metabolism; D010430:Pentosyltransferases / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology*; D014990:Xylans / Q000096:biosynthesis*
chemical_list: D005936:Glucans; D011993:Recombinant Fusion Proteins; D014990:Xylans; D049452:Green Fluorescent Proteins; C029353:xyloglucan; D005647:Fucosyltransferases; D005700:Galactosyltransferases; C478982:MUR3 protein, Arabidopsis; D010430:Pentosyltransferases