Identificazione e caratterizzazione di geni coinvolti nel processo di abscissione in frutti di melo (Malus domestica L. Borkh)

Many fruit species bear an abundance of flowers producing a surplus of fruits that the tree is unable to support. In anticipation of this, the major fruit species developed an immature fruit (fruitlet) physiological drop as a self-regulatory mechanism. This process is, at least in part, a consequence of the competition among fruits and between fruits and shoots for carbon assimilates. The self-regulatory mechanism responsible for the immature apple fruit shedding may be magnified by chemicals such as naphthaleneacetic acid (NAA) and its amide (NAD), and benzylaminopurine (BA) sprayed within 5-6 weeks after full bloom. The thinning action of bioregulators is quite variable and depends on environmental conditions and genotypes. In apple, there are varieties easy to thin and others difficult even though different chemicals or combinations of them are used. Understanding the molecular mechanisms and processes involved in abscission might help in finding new approaches and new chemical thinners to control abscission in fruit, or new self-thinning varieties. The described research was aimed to elucidate the molecular events underlying the in planta fruitlet abscission, taking into account the characteristics of this system and the practical importance of thinning in apple. Fruit drop is due to the activation of specific abscission zones (AZs). It is accepted that abscission is a highly regulated developmental process that is both influenced and activated in response to internal cues and/or environmental conditions. Nevertheless, the identity of the signals responsible for the activation of the AZ is as yet unknown. Among phytohormones, ethylene enhances abscission in several species and systems as well as in apple, while auxins produced by seeds are thought to desensitize AZs to ethylene and prevent abscission. In apple trees, the fruitlet physiological drop is due to the activation of the AZ located at the junction of the peduncle into the twig. In this region four lateral (LF) and one central (CF) fruitlets and the shoot are inserted. The CF comes from the pollinated king flower (KF) that, since it blooms earlier within the cluster, originates a fruitlet larger than the lateral ones. During the physiological drop, the shoot at cluster side, is thought to be a sink in competition with fruitlets for assimilate supply. Considering that seeds and/or fruits are involved in determining the shedding signal while the morphogenetic response occurs always at the AZ level, it is crucial to analyse the whole fruitlet system involved in abscission that should include concurrently seed, cortex, peduncle, and AZ. It is generally believed that the interaction between ethylene and auxin plays a major regulatory role in abscission. Starting from this, a mass gene approach was used in this work to identify genes regulating or involved in abscission. The cDNA-AFLP technique was adopted for transcriptional profiling of differentially expressed genes during apple fruitlet abscission. This allowed the isolation of 278 differential clones by comparing expression profiles of abscising (AF) versus non-abscising (NAF) fruitlet populations. AFs were obtained from lateral fruitlets of trees sprayed with benzylaminopurine (BA) at 200 ppm, 17 days after petal fall (APF) when the fruit cross diameter was about 10-12 mm. NAF originated from central flowers grown in clusters where all the lateral flowers had been removed at bloom. All ESTs (expressed sequence tags) obtained have been annotated with the Gene Ontology vocabulary and grouped according to cellular components, biological processes and molecular functions. Considering the cellular components, the most affected genes in the cortex were related to mitochondrion, plastid and membranes. Concerning the molecular functions, the mostly affected ones were the binding and the transferase activities in the cortex, the hydrolase and transport activities in the seed, and the binding activity in the peduncle. Considering the biological process, in the cortex the most abundant genes were those controlling transport, protein and carbohydrate metabolism. As a general remark, taking into account all the three ontology criteria, it appeared that a prominent up-regulation occurred in the seed. This might be consistent with the determinant role attributed to the seed in the regulation of fruit abscission. The expression and functional analyses of the most interesting clones were carried out by semiquantitative RT-PCR on agarose gel on cDNA obtained from seeds, cortex, peduncles and AZs of AFs and NAFs. Expression analyses confirmed the efficacy of the cDNA-AFLP approach to find a large amount of differentially expressed ESTs and the involvement of the studied genes in regulating the abscission and senescence processes. In particular the differential expression of sugar-metabolism and signalling related genes confirmed the importance of carbohydrates, together with hormones, in controlling the induction of AZs. Since functional studies through silencing or overexpression approaches cannot be easily performed on trees, additional experiments were carried out in Arabidopsis thaliana to investigate the participation of these and other genes in abscission. To this end, Arabidopsis genes putatively homologous to those differentially expressed in relation to fruitlet abscission in apple were identified. A dual approach was chosen to study their function in abscission. In a first attempt, insertional (T-DNA) homozygous mutants were obtained and scored for the presence of abscission-related phenotypes. Probably due to gene redundancy, no phenotypes were detected. Therefore, expression analyses were carried out on the same genes with real time RT-PCR on abscission zones of known Arabidopsis mutants with delayed (dab4-1, dab5-1) or no petal abscission (ida). The results showed a different pattern of expression in comparison to that found in wild type and confirmed an involvement of these genes in abscission. Current work is devoted to further characterise the putative role played by these genes in regulating the abscission of flower organs in Arabidopsis. In addition, a "systematic" approach for the analysis of the whole apple fruitlet abscission transcriptome is needed. To this end an apple microarray is being developed from the large number of already available ESTs, to be used for screening of new chemical thinners and for marker assisted selection of self thinning genotypes.