STUDY OF BIOCHEMICAL AND MOLECULAR MECHANISMS INVOLVED IN THE REGULATION OF RIPENING IN THE CLIMACTERIC PEACH (PRUNUS PERSICA L. BATSCH) FRUIT

Abstract Peach (Prunus persica[L.] Batsch) is temperate fruit species of economic importance, it ranks second in fruit production in Europe after apple. Peach fruit are classified by different flesh firmness, which strongly influences fruit shelf-life and storage, and ethylene evolution, which determinates the organoleptic quality of the ripening fruit. Concerning flesh texture, three main distinct peach fruit phenotypes are known, even if not all is understood in terms of their genetic determination and differences in biochemical pathways activated during the final stages of ripening which lead to the definition of phenotypic-specific characteristics. The first two phenotypes, described by Bailey and French, 1932, are the melting (M) and non-melting (NM) flesh. The M texture shows a prominent softening in the final stage of ripening. Variability of firmness is found within this phenotype and Yoshida (1976) distinguished between soft, medium and firm. The NM phenotype (the so called ‘canning peach’) shows a firm texture when fully ripe that never becomes soft. Rather, it becomes rubbery (for the loss of water from the tissues) during the senescence stage, when most cultivars could also display the development of peculiar off-flavours (Sherman et al., 1990). The third flesh texture phenotype was described by Yoshida (1976). He firstly classified a very firm and crispy, ‘stony hard’ (SH) flesh phenotype as belonging to the M family. However, these fruit never soften and resemble a NM phenotype, becoming rubbery when senescent. A possible fourth flesh texture trait resembles very much the SH flesh in firmness and crispiness, but when fully ripe becomes melting. This flesh texture is found in many recently developed new cultivars (nectarines, as ‘Big Top’, and standard peaches, e.g. ‘Rich Lady’ and ‘Diamond Princess’; Monet and Bassi, 2008). Availability of newly established peach cultivars with optimal organoleptic (as for M phenotypes) and durability [as for NM, SH and Slow Melting (‘Big Top’) phenotypes] characteristics would represent an economic advantage improving shelf-life and economic value of the produce. To do this, and in order to plan proper breeding programmes, a possible strategy may consist in the identification of molecular markers linked to the “flesh texture” phenotype. In this work, particular attention has been devoted to the development of different characteristics of flesh texture as a function of the structure of the endo-PG gene and of fruit ethylene evolution and response. The research work was articulated in four main topics: 1. Chapter I reports the results of some physiological, biochemical and molecular aspects of the Melting of the ‘Big Top’ Nectarine Fruit. We have characterized the postharvest behavior of ‘Big Top’ fruit, treated or not with ethylene for five days after harvest (DAH), and compared it with that of ‘193 Q XXVII 111’, a SH peach selection (‘Ghiaccio’), and with that of an accession with very firm flesh, ‘D41-62’. Pp-ACS1 expression, ethylene evolution, endo-PG production and softening characteristics have been evaluated in fruit of the three genotypes and referred to those of ripe melting flesh (M) ‘Bolero’ at harvest. Like ‘Bolero’, ‘Big Top’ fruit did express Pp-ACS1 and evolve ethylene, but with a 5-d delay during postharvest. Pp-endo-PG expression, production of an active endo-PG isoform typical of ripe peach fruits and fruit melting showed a parallel behavior; ethylene treatment further enhanced all the above features. In SH ‘Ghiaccio’ Pp-ACS1 expression, ethylene evolution, endo-PG production and softening were virtually absent during the first five DAH in air. ‘Ghiaccio’ neither expressed Pp-ACS1 nor evolved ethylene even after ethylene treatment, but responded by accumulating Pp-endo-PG transcripts and an active endo-PG protein, thus with consequent flesh melting. A similar behavior was observed in ‘D41-62’. Overall, the data confirm the pivotal role of ethylene in the regulation of endo-PG expression and activity, and thus in the determination of peach fruit flesh texture characteristics, and support the evidence that ‘Big Top’ could be classified as belonging to the ‘melting’ (‘slow melting’) phenotype and ‘D41-62’ to the SH one. 2. Chapter II describes the EndoPG locus configuration in different peach genotypes. The work has been undertaken with the aim of ascertaining whether specific mutations (SNPs or InDel polymorphisms, assessed by CAPS or InDel analysis) at the level of the Pp-endoPG gene could be used as molecular markers for prediction of fruit phenotype. To do so, a preliminary screening has been conducted on a few established cultivars on the progenies of a few selected crosses identifying, at the level of the endoPG gene, the patterns of InDel amplification and CAPS restriction patterns and comparing them with fruit texture (M, NM, SH, Slow Melting) phenotype. The results, albeit preliminary and to be confirmed on a larger number of selection from controlled crosses, seemed to provide satisfactory matching between the molecular results and fruit phenotype. 3. Chapter III reports the isolation and preliminary analysis of Pp-endoPG promoter region in Melting, Non Melting amd Slow Melting flesh genotypes. The sequences of the 5’ upstream region of the m Pp-endoPG clone of both NM ‘Oro A’ and M ‘Bolero’, of the M clone of M ‘Bolero and of the BT clone of Slow Melting ‘Big Top’ were obtained. PLACE analysis showed the presence in different positions of the isolated promoter sequences of a few interesting cis-acting elements, differently involved in the plant responses to endogenous (phytohormones) and exogenous (light, water stress, etc.) factors suggesting a possible fine regulation of Pp-endoPG expression by several factors. Particularly interesting was retrieval of microsatellites, possibly exploitable as molecular markers for the identification of the fruit phenotype, and of an Ethylene Responsive Element (ERE). The latter one was present in the 5’ upstream region of clones m-B and BT and in M clone, while in the 5’ upstream region of clone m-O a SNP was identified an altered ERE motif. This result may possibly explain the lower expression of Pp-endoPG observed in the high ethylene-producing NM cultivar ‘Oro A’. Preliminary gel Electrophoresis Mobility Shift Assay (EMSA) gave the indication of a stronger interaction with nuclear protein of the promoter region of clone m-B (containing ERE motif) with respect to clone m-O. This result, though needing to be confirmed by further experiments, appears consistent with the different expression profiles of the Pp-endoPG gene as related to softening behaviour 4. Chapter IV describes the preliminary characterization of some physiological and biochemical traits of fruit of one (BO 95021043) of the selections available at the Di.Pro.Ve., and of their response to treatment, in the postharvest period, with ethylene and/or with its antagonist, 1-methyl-1-cyclo-propene (1-MCP). The results obtained confirmed that these fruit were blocked in the evolution of all the ripening-related parameters. This physiological blockade was accompanied by an overall extremely low and constant degree of expression of a few genes playing a key role in ripening-related process. Lack of endogenous ethylene evolution in SR mutants seemed related to block of the expression of the Pp-ACO1 gene. SR fruit, though, were able to respond to ethylene treatment as demonstrated by the ethylene-induced slight enhancement of ripening-related parameters. Cleaved Amplified Polymorphis Sequence (CAPS) investigation on the structure of the Pp-endoPG gene in the SR BO 92051043 selection indicated the presence of two SNPs (SNP390 and SNP1310) common also to ‘Big Top’, ‘Ghiaccio’ and ‘Yumyeong’, possibly indicative of a parentage between these lines.