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

Marie Dumont, Arnaud Lehner, Boris Vauzeilles, Julien Malassis, Alan Marchant, Kevin Smyth, Bruno Linclau, Aurélie Baron, Jordi Mas Pons, Charles T. Anderson, Damien Schapman, Ludovic Galas, Jean Claude Mollet, Patrice Lerouge

Research output: Contribution to journalArticle

22 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)437-447
Number of pages11
JournalPlant Journal
Volume85
Issue number3
DOIs
StatePublished - Feb 1 2016

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Plant Cells
alkynes
Cell Wall
cell walls
image analysis
Acids
acids
dye binding
azides
Alkynes
fucose
monosaccharides
ligases
pectins
root growth
rhizosphere
cellulose
tobacco
chemical derivatives
copper

All Science Journal Classification (ASJC) codes

  • Genetics
  • Plant Science
  • Cell Biology

Cite this

Dumont, M., Lehner, A., Vauzeilles, B., Malassis, J., Marchant, A., Smyth, K., ... Lerouge, P. (2016). Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy- d -manno-oct-2-ulosonic acid. Plant Journal, 85(3), 437-447. https://doi.org/10.1111/tpj.13104
Dumont, Marie ; Lehner, Arnaud ; Vauzeilles, Boris ; Malassis, Julien ; Marchant, Alan ; Smyth, Kevin ; Linclau, Bruno ; Baron, Aurélie ; Mas Pons, Jordi ; Anderson, Charles T. ; Schapman, Damien ; Galas, Ludovic ; Mollet, Jean Claude ; Lerouge, Patrice. / Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy- d -manno-oct-2-ulosonic acid. In: Plant Journal. 2016 ; Vol. 85, No. 3. pp. 437-447.
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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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Dumont, M, Lehner, A, Vauzeilles, B, Malassis, J, Marchant, A, Smyth, K, Linclau, B, Baron, A, Mas Pons, J, Anderson, CT, Schapman, D, Galas, L, Mollet, JC & Lerouge, P 2016, 'Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy- d -manno-oct-2-ulosonic acid', Plant Journal, vol. 85, no. 3, pp. 437-447. https://doi.org/10.1111/tpj.13104

Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy- d -manno-oct-2-ulosonic acid. / Dumont, Marie; Lehner, Arnaud; Vauzeilles, Boris; Malassis, Julien; Marchant, Alan; Smyth, Kevin; Linclau, Bruno; Baron, Aurélie; Mas Pons, Jordi; Anderson, Charles T.; Schapman, Damien; Galas, Ludovic; Mollet, Jean Claude; Lerouge, Patrice.

In: Plant Journal, Vol. 85, No. 3, 01.02.2016, p. 437-447.

Research output: Contribution to journalArticle

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T1 - Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy- d -manno-oct-2-ulosonic acid

AU - Dumont, Marie

AU - Lehner, Arnaud

AU - Vauzeilles, Boris

AU - Malassis, Julien

AU - Marchant, Alan

AU - Smyth, Kevin

AU - Linclau, Bruno

AU - Baron, Aurélie

AU - Mas Pons, Jordi

AU - Anderson, Charles T.

AU - Schapman, Damien

AU - Galas, Ludovic

AU - Mollet, Jean Claude

AU - Lerouge, Patrice

PY - 2016/2/1

Y1 - 2016/2/1

N2 - 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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