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Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy- D-manno-oct-2-ulosonic acid

Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy- D-manno-oct-2-ulosonic acid
Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy- D-manno-oct-2-ulosonic acid
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.
0960-7412
437-447
Dumont, Marie
242cbdbb-c188-48d5-82b9-de124bdf8137
Lehner, Arnaud
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Vauzeilles, Boris
9a532ccd-34b3-4e06-a4b5-af6f8213a8f9
Malassis, Julien
4473f5cb-70c4-43f8-8389-9efe0a72e914
Marchant, Alan
3e54d51c-53b0-4df0-b428-2e73b071ee8e
Smyth, Kevin
320ba1b2-ce6e-4bbf-b3f3-243af5fd3e2f
Linclau, Bruno
19b9cacd-b8e8-4c65-af36-6352cade84ba
Baron, Aurelie
ecf2bfd2-949b-4cc1-bedf-505aadabc057
Mas Pons, Jordi
c792492b-64eb-4902-8e40-62a68c82c9c3
Schapman, Damien
48a90994-e04d-4b02-84ef-24fd969de19a
Galas, Ludovic
7916089e-1b40-47b2-ac43-022dc2020ec3
Mollet, Jean-Claude
1ea954fb-ff0a-4d43-aff6-8bc9d27bee30
Lerouge, Patrice
3e84085c-05d9-4e88-a6fe-1c112f104b85
Dumont, Marie
242cbdbb-c188-48d5-82b9-de124bdf8137
Lehner, Arnaud
112254e8-dea7-486e-a784-d4acb16463af
Vauzeilles, Boris
9a532ccd-34b3-4e06-a4b5-af6f8213a8f9
Malassis, Julien
4473f5cb-70c4-43f8-8389-9efe0a72e914
Marchant, Alan
3e54d51c-53b0-4df0-b428-2e73b071ee8e
Smyth, Kevin
320ba1b2-ce6e-4bbf-b3f3-243af5fd3e2f
Linclau, Bruno
19b9cacd-b8e8-4c65-af36-6352cade84ba
Baron, Aurelie
ecf2bfd2-949b-4cc1-bedf-505aadabc057
Mas Pons, Jordi
c792492b-64eb-4902-8e40-62a68c82c9c3
Schapman, Damien
48a90994-e04d-4b02-84ef-24fd969de19a
Galas, Ludovic
7916089e-1b40-47b2-ac43-022dc2020ec3
Mollet, Jean-Claude
1ea954fb-ff0a-4d43-aff6-8bc9d27bee30
Lerouge, Patrice
3e84085c-05d9-4e88-a6fe-1c112f104b85

Dumont, Marie, Lehner, Arnaud, Vauzeilles, Boris, Malassis, Julien, Marchant, Alan, Smyth, Kevin, Linclau, Bruno, Baron, Aurelie, Mas Pons, Jordi, Schapman, Damien, Galas, Ludovic, Mollet, Jean-Claude and Lerouge, Patrice (2016) Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy- D-manno-oct-2-ulosonic acid. The Plant Journal, 85 (3), 437-447. (doi:10.1111/tpj.13104). (PMID:26676799)

Record type: Article

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.

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Accepted/In Press date: 27 November 2015
e-pub ahead of print date: 30 January 2016
Published date: February 2016
Organisations: Centre for Biological Sciences

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Local EPrints ID: 391377
URI: http://eprints.soton.ac.uk/id/eprint/391377
ISSN: 0960-7412
PURE UUID: 7131fad8-b48f-4c15-83bb-d5155670c641
ORCID for Bruno Linclau: ORCID iD orcid.org/0000-0001-8762-0170

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Date deposited: 12 Apr 2016 09:23
Last modified: 17 Dec 2019 01:52

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Contributors

Author: Marie Dumont
Author: Arnaud Lehner
Author: Boris Vauzeilles
Author: Julien Malassis
Author: Alan Marchant
Author: Kevin Smyth
Author: Bruno Linclau ORCID iD
Author: Aurelie Baron
Author: Jordi Mas Pons
Author: Damien Schapman
Author: Ludovic Galas
Author: Jean-Claude Mollet
Author: Patrice Lerouge

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