Proteomic comparative analysis to study the transition from the pre-climacteric to the climacteric stage in two peach fruit cultivars

The ripening process in fruit is a complex phenomenon during which sharp metabolic changes occur (i.e. chlorophyll degradation and pigments biosynthesis, simple sugars and organic acids accumulation, volatiles production and flesh softening). All these events are genetically.programmed but also influenced by the environment. Climacteric fruit, which comprise economically-relevant species like tomato and peach, are characterized by a peculiar surge in ethylene evolution and respiration rate at the onset of ripening. The role of ethylene has been largely studied and it has been demonstrated to be involved in many of the processes mentioned above. ACC oxidase (ACO) catalyses ethylene production from ACC (1-amynocyclopropane-1-carboxilic acid) and the expression levels of this enzyme have extensively been proved to mirror ethylene evolution. According to their flesh phenotypes at ripening, peach (Prunus persica [L.] Batsch) fruit are classified as either melting flesh (MF) and non melting (NMF): they both soften at maturity but this event is more evident in ripe MF than in NMF fruits. For this reason, the two cultivars have different market destinations: MF fruits, which are soft and juicy at harvest, are typically destined to fresh consumption; NMF peaches, on the contrary, are mainly destined to canning since they have good keeping qualities. We applied the proteomic approach to study the transition from the pre-climacteric to the climacteric phase in Bolero, a MF cultivar, and in Oro A, a typical NMF cultivar. The different samples were established by a Western blot analysis using anti-ACC oxidase antibodies. Mesocarp proteins from drupes at the pre-climacteric and climacteric stages of the cv Bolero (MF) and Oro A (NMF) were resolved by 2D-PAGE on a linear pH 3-10 gradient and 24 cm 12.5% polyacrylamide gels. Gel images and statistical analyses, conducted with ImageMaster Platinum 5.0 and the ANOVA test (P<0.01) respectively, showed that 53 spots had a significant 2-fold expression change. By means of LC-ESI-MS/MS the spots of interest were characterized. The spots were grouped according to their expression trends during the transition from the pre-climacteric to the climacteric phase. Among the proteins differentially expressed in both cultivars at the climacterium, five of them belonged to the ethylene biosynthetic pathway: we identified two forms of ACO (according to the literature reporting a dramatic increase in ethylene evolution at the climacterium), two isoforms of S-adenosyl-methionine synthase and a spot referring to _β-cyanoalanine synthase, the enzymes involved in ethylene biosynthesis and detoxification of cyanide, a by-product of ethylene evolution. Some spots were identified as proteins involved in the amino acid and carbon metabolisms as well as in the translation process. Nevertheless some identified proteins showed a different trend between the two genotypes, thus suggesting that peculiar events characterize the ripening process in the two cultivars.