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, At3g57690 5'-AAGAAGATTGCTTGCGGTGTGC-3' 5'-AAAGAGCCAAGAGCTGGCAACG-3'

, Nom Amorce sens Amorce antisens sia2-1 5'-GCAAATGGTTTGGGACTACAA-3' 5'-TGTTTCAGGAAGCACCAATG-3'

, Calcium-and magnesium-free Dulbecco's phosphate-buffered saline (CMF-DPBS) (see Recipes)

, Olympus CK2 inverted microscope 3. Microscope Zeiss AxiObserver Z1

, Syringe Filter PVDF 33 mm 0.2 µm 5. Microcentrifuge (VWR TM Galaxy 14D)

, NucleoSpin® Plasmid Binding Columns (Macherey-Nagel, catalog number: 740588.50), but columns of any other kits (Genejet or PureLink) may be used

, nm; emission, 520-560 nm wavelengths Procedure Note: All transfer and all washing steps described below are performed with a tip cut 0.5 cm from its extremity to avoid pollen tube damages, Confocal Microscope TCS-SP2 operating system (Leica) and microscope Leica DMI6000B with a DFC450 C camera with FITC filter (absorption, pp.485-520

, US/Shopping/ProductDetails.aspx?productid=T-205-WB

, Remove the medium by aspiration or centrifugation and wash three times with 100 µl TBS buffer (or CMF-DPBS, both filtered at 0.22 µm using a syringe filter). Pollen tubes are gently resuspended in the buffer by pipetting up and down and the buffer is removed by aspiration/centrifugation

, Seal the bottom of the column with parafilm ® and add 100 µl 0.002% saponin in TBS for 15 min for pollen membrane permeabilization (skip this step and the next one for the cell wall immunolabeling)

, Seal the bottom of the column with parafilm ® and incubate the pollen tubes with 100 µl blocking buffer 1 (or blocking buffer 2), freshly prepared

, The cell wall labelling requires three washes with CMF-DPBS as explained in step B3 (Skip this step for the membrane immunolabelling)

, Seal the bottom of the column and add 50 µl of primary antibody 1:10 dilution DGDG-specific rabbit serum in TBS-1%FBS-0.002% saponin and incubate 1 h at room temperature (or for the cell wall labelling: 1:20 primary antibody in CMF-DPBS overnight at 4 °C)

, Wash three times with TBS-1% FBS-0.002% saponin (or CMF-DPBS) as explained in step B3

, Seal the bottom of the column and add 50 µl 1:500 dilution Alexa secondary antibody in TBS1%FBS-0.002% saponin and incubate 1 h at room temperature (or for the cell wall labelling: FITC-coupled secondary antibody 1:50 dilution in CMF-DPBS for 2 h at 30 °C)

, Note: From this step, keep the tubes in the dark in aluminum foil

, TBS-1% FBS-0.002% saponin and a final wash with TBS (or five times with CMF-DPBS) as explained in step B3

, resuspend them by adding at the top of the column 100 µl of TBS (or water) and by pipetting up and down several times with an enlarged tip and transfer the solution which contains the pollen tubes on a microscope slide with a cover slip. Figure 3 shows the confocal images showing the results of immunofluorescence labelling of Arabidopsis pollen tubes with anti-DGDG. Figure 4 gives an overview of the method for the membrane or cell wall immunolabelling of pollen tubes

, Calcium-and magnesium-free Dulbecco's phosphate-buffered saline (CMF-DPBS)

L. C. Boavida and S. Mccormick, TECHNICAL ADVANCE: Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana, Plant J, vol.52, issue.3, pp.570-582, 2007.

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URL : https://hal.archives-ouvertes.fr/hal-01805112

M. Dumont, A. Lehner, S. Bouton, M. C. Kiefer-meyer, A. Voxeur et al., The cell wall pectic polymer rhamnogalacturonan-II is required for proper pollen tube elongation: implications of a putative sialyltransferase-like protein, Ann Bot, vol.114, issue.6, pp.1177-1188, 2014.
URL : https://hal.archives-ouvertes.fr/hal-01204135

T. Matoh, M. Takasaki, K. Takabe, and M. Kobayashi, field: Plant Science > Plant biochemistry > Carbohydrate Category by field: Biochemistry > Carbohydrate > Polysaccharide Category by organism: Plants > Arabidopsis > Pollen >carbohydrate Analysis of Sugar Component of a Hot Water, Plant Cell Physiol, vol.39, pp.483-491, 1998.

F. Aignan, COBRA, UMR 6014 et FR 3038

. Abstract, Extraction with hot water is the oldest and simplest method used to recover pectin from an alcohol insoluble residue extract, although this method has not been widely used for the cell wall analysis of pollen tube, a model used to study cell wall

, Arabidopsis day-0 flowers according to Boavida and McCormick, 2007.

, Absolute ethanol for analysis (Merck Millipore, p.107017

, Trifluoroacetic acid 12.93N (TFA) (Sigma-Aldrich Reagent Plus®, catalog number: T62200)

, catalog number: I5125), vol.2

, Methanol (Fisher chemical, p.10010280

, Silylation reagent (HMDS:TMCS:Pyridine, 3:1:9 Sylon? HTP), p.33038

, Cyclohexane (Acros Organics, p.279590010

, Arabidopsis pollen germination medium (see Recipes)

A. Monosaccharides-standards,

(. Fucose and . Sigma-aldrich,

(. Galactose and . Sigma-aldrich, catalog number G0750) Galacturonic acid, p.857289

, Glucose (Merck Millipore, catalog number 8337.0250) Glucuronic acid

(. Mannose and . Sigma-aldrich,

(. Rhamnose and . Sigma-aldrich,

(. Xylose and . Sigma-aldrich, catalog number X2126)

, Equipment 1. Inverted microscope Olympus CK2

C. Allegra and ®. Coulter, Hot-water bath (70 °C and 90 °C) 4. 40-ml glass potter homogenizer 5. Freeze-dryer 6, vol.3

, Sample concentrator under air flow (Techne, catalog number: FSC400D)

G. , instrument is composed of a Zebron Z5-MSi (30 m, 0.25 mm id, 0.25 µm film thickness, Phenomenex) capillary column for chromatographic separations, a Hewlett-Packard 6890 series gas chromatograph coupled to an Autospec mass spectrometer of EBE geometry

, Microliter syringe (10 µL

, Grow in vitro Arabidopsis pollen according to the method described by, 2007.

, Harvest 40 freshly open Arabidopsis thaliana flowers (morning is the best)

, Submerge in 1 ml of pollen germination medium in a 1.5 ml Eppendorf tube

L. C. Boavida and S. Mccormick, TECHNICAL ADVANCE: Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana, Plant J, vol.52, issue.3, pp.570-582, 2007.

M. Dumont, A. Lehner, S. Bouton, M. C. Kiefer-meyer, A. Voxeur et al., The cell wall pectic polymer rhamnogalacturonan-II is required for proper pollen tube elongation: implications of a putative sialyltransferase-like protein, Ann Bot, vol.114, issue.6, pp.1177-1188, 2014.
URL : https://hal.archives-ouvertes.fr/hal-01805177

, Cuisine du monde, Voyages

. Directeurs-de-thèse, The Pennsylvania State University-Biology Department (Anderson CT lab)USA « Click Chemistry and Fucosyltransferase inhibition combination ». 2012 : Mémoire de recherche, Laboratoire Glyco-MEV-EA 4358-Université de Rouen « Immunité végétale : caractérisation des Arabinogalactanes protéines de Fabacées et effets sur les oomycètes pathogènes», Soutenance prévue : juillet 2015. 2013 : Séjour scientifique, 2011.

M. Dumont, A. Lehner, S. Bouton, M. C. Kiefer-meyer, A. Voxeur et al., The cell wall pectic polymer rhamnogalacturonan-II is required for proper pollen tube elongation: implications of a putative sialyltransferase-like protein. IASPRR (International Association of Sexual Plant Reproduction Research, Juillet, pp.13-18, 2014.
URL : https://hal.archives-ouvertes.fr/hal-01805177

M. Dumont, A. Voxeur, A. Lehner, J. C. Mollet, and P. Lerouge, Role of Rhamnogalacturonan type II in pollen tube elongation, XIII th Cell Wall Meeting, pp.7-12, 2013.
URL : https://hal.archives-ouvertes.fr/hal-02119181

M. Dumont, A. Voxeur, A. Lehner, J. C. Mollet, and P. Lerouge, Role of Rhamnogalacturonan type II in pollen tube elongation. 2 nd Journée de l'Institut de Recherche et d'Innovation Biomédicale, 2013.
URL : https://hal.archives-ouvertes.fr/hal-02119181