MALDI-ToF Ms Analysis Of Plants Treated with Acybenzolar-S Methyl.

Acybenzolar-S-methyl (ASM) is a synthetic inducer and a functional analogue of salicylic acid, involved in systemic acquired resistance (SAR). In our study, we assessed the expression of proteins, in Solanum lycopersicum. leaves treated with ASM. Tomato leaves were gound into a fine powder with liquid nitrogen and stored at -70°C. For protein extraction, the powder was washed twice with ethanol and deionized water, after centrifugation the final pellets were dried in speed-vac and dissolved in 1% (w/v) SDS, 20% (v/v) glycerol, 40 mM DTT, 50 mM Tris-HCl, pH 7.5. Protein content was determined according to the method of Bradford. Aliquots of extracts from untreated and ASM-treated samples were precipitated with chloroform/methanol and the protein pellets were dried and analyzed by 2-D electrophoresis. IEF (Isoelectrophocusing) steps were performed on IPG dry-strips of 13 cm in non-linear pH gradient of 3-11 (GE-Heathcare), proteins were separated in the 2nd dimension (SDS-PAGE) in a 13% (w/v) polyacrylamide gel. Gels were coomassie stained and analyzed by the ImageMaster 2D Elite software (Amersham Biosciences). Three gels were produced for untreated and ASM-treated samples, respectively. Protein spots present on at least two gels were used for comparison of the obtained spots. Image analysis of 2-DE gels revealed that 116±8 and 124±11 spots were extracted from control and ASM-treated samples, respectively. Proteins were considered differentially expressed when spot intensities (normalized volume) varied of at least a two-fold differences (ANOVA; P < 0.05) in up or down regulation respect to the untreated sample (control). Protein spots that significantly changed in response to the ASM-treatment with respect to the control, were successfully identified by MALDI-ToF analysis. Twentyeight spots identified as corresponding to 17 different proteins were classified in three groups. on the basis of their function, Of these, the majority (18 spots) were involved in defense mechanisms/ stress responses, seven spots in photosynthetic metabolism and three spots as proteins associated to energy metabolism