Proteomic analysis of β-1,3-glucanase in grape berry tissues

Grape berries are considered recalcitrant materials in proteomic analysis, because berry tissues contain large amounts of secondary metabolites, especially phenolic compounds, which severely interfere with protein extraction and electrophoresis separation. We report hereby a PVPP/TCA-based protein extraction protocol for grape berries. Phenolic compounds in berry extracts were removed with repeated PVPP cleanups, and proteins were recovered with TCA precipitation. Protein resolution in 2-D gels was gradually improved with the increase of PVPP cleanup steps. By the protocol, about 760 protein spots of berry tissues were clearly resolved in 2-D gels with CBB staining. This protocol was also used to analyze β-1,3-glucanase (EC 3.2.1.39) in berry tissues. An anti-synthetic peptide antibody was prepared against 15 amino acid sequence residing on the surface of β-1,3-glucanase molecule. It detected two major spots in 2-D blots of berry extracts. The spots were identified by MALDI-TOF analysis as β-1,3-glucanase. The present study validates that β-1,3-glucanase is present in higher abundance in berry skins than in pulps, and in red berries than in white berries. Therefore, β-1,3-glucanase displays a tissue-specific expression. The preferential accumulation of β-1,3-glucanase in skins may be relevant to berry ripening. © 2009 Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków.