INDIVIDUAZIONE DI INDICATORI DI QUALITÀ E MIGLIORAMENTO DEL PROCESSO PRODUTTIVO IN DRUPACEE (PESCO) MEDIANTE METODOLOGIE PROTEOMICHE INNOVATIVE

Fruit ripening is a complex process genetically determined and environmentally regulated that involves drastic changes in various physiological and biochemical aspects. These events include chlorophyll breakdown, increased starch degradation and simple sugar biosynthesis, development of aroma components and fruit softening. All these events have a direct impact on the definition of fruit quality and are accompanied and/or at least partially influenced by less visible processes like ethylene evolution and respiration. These phenomena are typical of a class of fruits like tomato, banana and peach (the so-called climacteric fruits). In particular, peach fruit development follows a sigmoidal curve divided into four stages [S1, cell division and expansion; S2, pit hardening and slowdown in fruit growth; S3, increase in fruit size due to cell enlargement; S4, ethylene production and increased respiration (climacteric peak)]. Afterwards the ripening process comes to an end and fruits start senescing. The transition from the pre-climacteric to the climacteric phase is a critical step for fruit development and has been studied with a transcriptomic approach in peach fruit by means of the first available fruit microarray gene chip μpeach 1.0. The aim of the first part of this PhD research was the study of this transition in peach fruit with a proteomic approach. Peach is a typical climacteric fruit whose rapid softening in postharvest makes it particularly susceptible to handling and manipulation. Actually, different flesh firmness phenotypes at maturity are known. Most of the economically relevant peach varieties are divided into melting flesh (MF) and non melting flesh (NMF) phenotypes: they both soften but this event is more relevant in MF than in NMF cultivars. This behaviour makes MF peaches soft and juicy, and particularly appreciated by the consumers but shortens their shelf life, while NMF peaches have good keeping qualities but are less appreciated for fresh consumption. In order to perform the proteomic analysis proteins from freeze-dried mesocarp samples at S3 and S4 ripening stages of the cv Bolero (MF) and cv Oro A (NMF) were resolved by 2D PAGE on a linear 3-10 pH range and 24 cm 12.5% polyacrylamide gels. Gel image and statistical analyses, conducted with ImageMaster Platinum 5.0 and the ANOVA test (p<0,01), respectively, showed that 53 spots had a statistically relevant 2-fold expression change. By means of LC-ESI MS/MS we found that some proteins were involved in different physiological processes (i.e. sugar metabolism, ethylene evolution, amino acid metabolisn and stress response) typical of fruit development and ripening. These data, however, describe only a short moment of a complex and very long process like fruit ripening. For this reason, the second part of this PhD research focused on the comparative proteomic analysis of a complete growth curve of a single cultivar, cv Springcrest (MF). Drupes of five ripening stages (S1, S2, S3, S4 I and S4 II), established according to the literature, were sampled in the period April-June 2008 and protein extraction and separation by 2D-PAGE were performed after the optimization of the extraction protocol. Mesocarp proteins were resolved by basic loading of 400 micrograms onto a linear 4-7 pH range 24 cm Immobiline dry strips followed by SDS-PAGE on 10% polyacrylamide gels. In silico and statistical analyses, conducted with ImageMaster Platinum 5.0 and the ANOVA test (p<0,001), respectively, showed that 98 spots had a statistically relevant 2-fold expression change. The hierarchical clustering analysis grouping the 98 variations revealed that, while S1 and S2 are quite similar and the S4I and S4II as well, S3 shows a peculiar and totally different behaviour when compared to the first and last two ripening stages. The mass spectrometry analysis aiming at the identification of the differentially expressed proteins is now in progress.