Unravelling the transition from the pre-climacteric to the climacteric stage in peach fruit : a comparison between cultivars

Background: Fruit ripening is a very complex phenomenon, since it is regulated by a huge number of genes and is also subjected to environmental influence. Many important metabolic events take place during this process, the most remarkable of which are chlorophyll degradation and pigments biosynthesis, simple sugars and organic acids accumulation, volatiles production and flesh softening. According to the patterns of ethylene evolution and respiration, fruits are classified as climacteric and non climacteric. The latter show a gradual decline in respiration all throughout the ripening process, while the former, 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 physiological processes mentioned above. Among the enzymes of the biosynthetic pathway, the levels of ACC oxidase (ACO), which catalyzes the conversion of 1-aminocyclopropane-1-carboxylic acid into ethylene, have been proved to mirror ethylene evolution. According to their flesh phenotypes at ripening, peach (Prunus persica [L.] Batsch) fruits are grouped into melting flesh (MF) and non melting flesh (NMF): they both soften but this event is more evident in mature MF than in NMF fruits. This behaviour also has economical consequences since it makes MF fruits soft and juicy and consequently attractive to the consumers but also extremely susceptible to handling and physical injuries; NMF peaches, on the contrary, are less appreciated by the consumers but have good keeping qualities. In this work we applied the proteomic approach to study some biochemical and physiological features of peach fruit ripening by comparing the protein profiles of two peach cultivars with different flesh firmness characteristics, sampled immediately before and during the climacterium. Methods: At least 15 fruits of the cv Bolero (MF) and Oro A (NMF) at the pre-climacteric and climacteric stages were sampled. The different samples were established by a Western blot analysis using anti-ACO antibodies. The mesocarp fraction of every ripening stage was isolated and total proteins from freeze-dried powder were directly extracted with phenol after setting up an adequate extraction protocol. Proteins werere solved by 2D-PAGE on a 24 cm linear pH 3-10 gradient and 12.5% polyacrylamide gels; gels were stained with colloidal Coomassie Brilliant Blue G-250. Gel images and statistical analyses were conducted with ImageMaster Platinum 5.0 and the ANOVA test (P<0.01) respectively. By means of LC-ESI-MS/MS the spots of interest were finally characterized. Results: Preliminary experiments were focalized to optimize the extraction procedure for peach mesocarp tissue.Gel analyses revealed 53 spots having a statistically significant and 2-fold expression change; 43 among them were analyzed. The spots were grouped according to their expression trends during the transition from the pre-climacteric to the climacteric phase: among the proteins up-regulated in both cultivars at the climacterium we identified two isoforms of ACO. Among the down-regulated spots we isolated Rubisco, two isoforms of S-adenosyl methionine synthase (MAT1 and MAT2) and beta-cyanoalanine synthase. Moreover we found some enzymes involved in the detoxification of Reactive Oxygen Species (ROS): a catalase whose expression levels were decreasing during ripening and a superoxide dismutase which, on the other hand, increased with the climacterium. Finally we identified a malate dehydrogenase cultivar specific, since it was only present in cv Bolero and absent in cv Oro A. Conclusions: The extraction procedure set up in this work allowed to obtain high quality, reproducible protein patterns, as confirmed by the gel comparison and the hierarchical clustering analyses.The expression trends of the spots referring to proteins directly involved in the ethylene biosynthetic pathway were in agreement with the literature: we found two forms of ACO, one of which was particularly abundant (it represented 2% of the total relative spot volume in the climacteric pattern of cv Bolero) displaying a dramatic increase in ethylene evolution at the climacterium. Moreover, we found a beta-cyanoalanine synthase, which is responsible for the detoxification of cyanide, a by-product of ethylene evolution. Surprisingly, between the two isoforms of S-adenosyl methionine synthase, MAT2 decreased in both cultivars, while a decrease of MAT1 was found in cv Bolero. Moreover, both MAT1 and MAT2 were less expressed in cv Oro A than in cv Bolero. This feature could be due to the different genetic traits of the two cultivars but also reflect a different ripening trend. Nevertheless, this apparent discrepancy may be linked to different roles of S-adenosylmethionine. This metabolite, in fact, is involved as methyl donor in a plethora of other biosynthetic processes than ethylene production (e.g. DNA methylation). During ripening fruit turns from green, where photosynthesis is still active, into a sink organ in which simple sugars are accumulated. According to this event we observed a decrease in a spot corresponding to Rubisco and an increase in a sucrose synthase levels. The ripening process has also been defined as a protracted form of senescence: we found three spots referring to proteins involved in the detoxification of ROS with different expression trends during the preclimacterium-climacterium transition. Two spots corresponding to a catalase and a peroxidase decreased while a superoxide dismutase increased during the transition, suggesting that the ripening process is characterized by a modulation of the different antioxidant enzymes, some of which are also involved in the synthesis of secondary compounds.