Development of a method for the extraction and analysis of grape skin proteins strongly bound to cell walls
2013
Pasquier, Grégory | Lapaillerie, Delphine | Dupuy, Jean-William | Lomenech, Anne-Marie | Claverol, Stéphane | Bonneu, Marc | Geny, Laurence | Teissedre, Pierre-Louis | Donèche, Bernard
Aim: To better understand the protein composition of grape skin cell walls, we have developed a method to analyse the strongly bound cell wall proteins. Methods and results: The protocol was developed with grape skins at full maturity. The critical steps of this protocol were : (i) the elimination of cellular aggregates, (ii) the elimination of soluble proteins, and (iii) the localization of the identified proteins within the cell wall. To verify whether these three conditions were met, the decrease in the quantity of cellular aggregates was followed by optical microscopy, the removal of soluble proteins was measured by chemical assay, and the presence of proteins located in cell walls was demonstrated by extensive bioinformatic analysis. The process made it possible to obtain a four-fold reduction in the amount of cellular aggregates, a reduction in the concentration of soluble proteins below the method detection limit, and a high proportion of proteins predicted to be secreted (79 %). Conclusion: The protocol described in this paper constitutes the first method to analyse proteins strongly bound to cell walls in grape skins. However, this method excludes the identification of labile proteins or proteins weakly bound to the cell wall. Significance and impact of the study: This protocol can be used for studying the role that strongly bound cell wall proteins play in development and defense processes in grape skins.
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