No abstract

An efficient protein extraction method is crucial to ensure successful separation by two-dimensional electrophoresis (2-DE) for recalcitrant plant species, in particular for grapevine (Vitis vinifera L.). Trichloroacetic acid-acetone (TCA-acetone) and phenol extraction methods were evaluated for proteome analysis of leaves and roots from the Tunisian cultivar 'Razegui'. The phenol-based protocol proved to give a higher protein yield, a greater spot resolution, and a minimal streaking on 2-DE gels for both leaf and root tissues compared with the TCA-based protocol. Furthermore, the highest numbers of detected proteins on 2-DE gels were observed using the phenol extraction from leaves and roots as compared with TCA-acetone extraction.

Proteomic and transcriptomic analyses of pathogenesis-related proteins, PR10, were carried out by subjecting adult plants of the salt-tolerant grapevine cv. Razegui to 100 mM NaCl for 6 and 24 h under controlled greenhouse conditions. Within 6 h of salt exposure, an increase in levels of RzPR10 was observed in the roots. PR10 mRNA levels in leaves were significantly different than those in roots; moreover, significant differences for mRNA levels were found between treated and control plants. Within 24 h of salt treatment, roots exhibited the highest levels of RzPR10 protein, but no differences were observed for mRNA levels in both leaves and roots. Overexpression of Vvpr10 cDNA, under the control of the Gal promoter, in the yeast Saccharomyces cerevisiae revealed that growth of transformed cells on a minimal medium containing 500 mM NaCl is similar to that of cells cultivated on NaCl-free medium. This indicated that Vvpr10 gene was functional in yeast and conferred tolerance to high salt concentrations.

Several Scanning microscopy techniques were used to investigate the leaf surface ultrastructure in the local "Razegui" grapevine cultivar (Vitis vinifera L.). Conventional scanning electron microscopy performed on glutaraldehyde-fixed samples allowed observation of well-preserved epidermal cells with an overlaying waxy layer. At a high magnification, the waxy layer exhibited crystalline projections in the form of horizontal and vertical platelets. Also, to avoid eventual ultrastructural alterations inherent in the use of solvents during sample preparation, fresh leaf blade samples were directly observed by environmental scanning electron microscopy. A classical image of convex living epidermal cells was observed. At 2400x magnification, epicuticular waxes exhibited a granular structure. However, high-magnification images were not obtained with this device. The atomic force microscopy (AFM) performed on fresh leaf blade samples allowed observation of a textured surface and heterogeneous profiles attributed to epicuticular wax deposits. AFM topography images confirmed further, the presence of irregular crystalloid wax projections as multishaped platelets on the adaxial surface of grapevine leaf.