title: Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy-D-manno-oct-2-ulosonic acid.
abstract: In plants, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) is a monosaccharide that is only found in the cell wall pectin, rhamnogalacturonan-II (RG-II). Incubation of 4-day-old light-grown Arabidopsis seedlings or tobacco BY-2 cells with 8-azido 8-deoxy Kdo (Kdo-N3 ) followed by coupling to an alkyne-containing fluorescent probe resulted in the specific in muro labelling of RG-II through a copper-catalysed azide-alkyne cycloaddition reaction. CMP-Kdo synthetase inhibition and competition assays showing that Kdo and D-Ara, a precursor of Kdo, but not L-Ara, inhibit incorporation of Kdo-N3 demonstrated that incorporation of Kdo-N3 occurs in RG-II through the endogenous biosynthetic machinery of the cell. Co-localisation of Kdo-N3 labelling with the cellulose-binding dye calcofluor white demonstrated that RG-II exists throughout the primary cell wall. Additionally, after incubating plants with Kdo-N3 and an alkynated derivative of L-fucose that incorporates into rhamnogalacturonan I, co-localised fluorescence was observed in the cell wall in the elongation zone of the root. Finally, pulse labelling experiments demonstrated that metabolic click-mediated labelling with Kdo-N3 provides an efficient method to study the synthesis and redistribution of RG-II during root growth.
authors.affiliation: Laboratoire Glycobiologie et Matrice Extracellulaire Végétale (Glyco-MEV), EA 4358, IRIB, VASI, Normandie Université, 76821, Mont-Saint-Aignan, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Dumont
authors.affiliation: Laboratoire Glycobiologie et Matrice Extracellulaire Végétale (Glyco-MEV), EA 4358, IRIB, VASI, Normandie Université, 76821, Mont-Saint-Aignan, France.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Lehner
authors.affiliation: Institut de Chimie des Substances Naturelles (ICSN) UPR CNRS 2301, 91198, Gif-sur-Yvette, France.|Institut de Chimie Moléculaire et des Matériaux d'Orsay (ICMMO) UMR CNRS 8182, Université de Paris Sud, 91405, Orsay, France.|Click4Tag, Zone Luminy Biotech, Case 922, 163 Avenue de Luminy, 13009, Marseille, France.
authors.forename: Boris
authors.initials: B
authors.lastname: Vauzeilles
authors.affiliation: Chemistry, University of Southampton, Southampton, SO17 1BJ, UK.
authors.forename: Julien
authors.initials: J
authors.lastname: Malassis
authors.affiliation: Centre for Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK.
authors.forename: Alan
authors.initials: A
authors.lastname: Marchant
authors.affiliation: Chemistry, University of Southampton, Southampton, SO17 1BJ, UK.
authors.forename: Kevin
authors.initials: K
authors.lastname: Smyth
authors.affiliation: Chemistry, University of Southampton, Southampton, SO17 1BJ, UK.
authors.forename: Bruno
authors.initials: B
authors.lastname: Linclau
authors.affiliation: Institut de Chimie des Substances Naturelles (ICSN) UPR CNRS 2301, 91198, Gif-sur-Yvette, France.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Baron
authors.affiliation: Institut de Chimie des Substances Naturelles (ICSN) UPR CNRS 2301, 91198, Gif-sur-Yvette, France.
authors.forename: Jordi
authors.initials: J
authors.lastname: Mas Pons
authors.affiliation: Department of Biology and Center for Lignocellulose Structure and Formation, The Pennsylvania State University, University Park, PA, USA.
authors.forename: Charles T
authors.initials: CT
authors.lastname: Anderson
authors.affiliation: PRIMACEN, IRIB, Normandie Université, 76821, Mont-Saint-Aignan, France.
authors.forename: Damien
authors.initials: D
authors.lastname: Schapman
authors.affiliation: PRIMACEN, IRIB, Normandie Université, 76821, Mont-Saint-Aignan, France.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Galas
authors.affiliation: Laboratoire Glycobiologie et Matrice Extracellulaire Végétale (Glyco-MEV), EA 4358, IRIB, VASI, Normandie Université, 76821, Mont-Saint-Aignan, France.
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Mollet
authors.affiliation: Laboratoire Glycobiologie et Matrice Extracellulaire Végétale (Glyco-MEV), EA 4358, IRIB, VASI, Normandie Université, 76821, Mont-Saint-Aignan, France.
authors.forename: Patrice
authors.initials: P
authors.lastname: Lerouge
mesh_terms: D017360:Arabidopsis / Q000648:ultrastructure*; D001386:Azides / Q000737:chemistry; D002473:Cell Wall / Q000648:ultrastructure*; D002478:Cells, Cultured; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors*; D010368:Pectins / Q000737:chemistry*; D018517:Plant Roots / Q000648:ultrastructure; D036226:Seedlings / Q000648:ultrastructure; D013194:Staining and Labeling; D013400:Sugar Acids / Q000737:chemistry*; D014026:Nicotiana / Q000648:ultrastructure
chemical_list: C000608137:8-azido-3,8-dideoxyoctulosonate; D001386:Azides; D013400:Sugar Acids; C042491:rhamnogalacturonan I; C042492:rhamnogalacturonan II; C002532:2-keto-3-deoxyoctonate; D010368:Pectins; D009713:Nucleotidyltransferases; C028984:3-deoxy-manno-octulosonate cytidyltransferase
keywords: Arabidopsis; BY-2 cells; Kdo-N3; cell wall; click chemistry; rhamnogalacturonan-II; technical advance