Molecular Studies of Postharvest Leaf and Flower Abscission

Original objectives: Understanding the regulation of abscission competence by exploring the nature and function of auxin-related gene expression changes in the leaf and pedicelAZs of tomato (as a model system), was the main goal of the previously submitted proposal. We proposed to achieve this goal by using microarray GeneChip analysis, to identify potential target genes for functional analysis by virus-induced gene silencing (VIGS). To increase the potential of accomplishing the objectives of the previously submitted proposal, we were asked by BARD to show feasibility for the use of these two modern techniques in our abscission system. Thus, the following new objectives were outlined for the one-year feasibility study: 1.to demonstrate the feasibility of the VIGS system in tomato to perform functional analysis of known abscission-related genes; 2. to demonstrate that by using microarray analysis we can identify target genes for further VIGS functional analysis. Background to the topic: It is a generally accepted model that auxin flux through the abscission zone (AZ) prevents organ abscission by rendering the AZ insensitive to ethylene. However, the molecular mechanisms responsible for acquisition of abscission competence and the way in which the auxin gradient modulates it are still unknown. Understanding this basic stage of the abscission process may provide us with future tools to control abscission for agricultural applications. Based on our previous study, performed to investigate the molecular changes occurring in leaf and stem AZs of MirabillisJalapaL., we have expanded our research to tomato, using genomic approaches that include modern techniques for gene discovery and functional gene characterization. In our one-year feasibility study, the US team has established a useful system for VIGS in tomato, using vectors based on the tobacco rattle virus (TRV), a Lcreporter gene for silencing (involved in regulation of anthocyanin biosynthesis), and the gene of interest. In parallel, the Israeli team has used the newly released Affymetrix Tomato GeneChip to measure gene expression in AZ and non-AZ tissues at various time points after flower removal, when increased sensitivity to ethylene is acquired prior to abscission (at 0-8 h), and during pedicelabscission (at 14 h). In addition, gene expression was measured in the pedicel AZ pretreated with the ethylene action inhibitor, 1-methylcyclopropene (1-MCP) before flower removal, to block any direct effects of ethylene. Major conclusions, solutions and achievements: 1) The feasibility study unequivocally established that VIGS is an ideal tool for testing the function of genes with putative roles in abscission; 2) The newly released Affymetrix Tomato GeneChip was found to be an excellent tool to identify AZ genes possibly involved in regulation and execution of abscission. The VIGS-based study allowed us to show that TAPG, a polygalacturonase specifically associated with the tomato AZ, is a key enzyme in the abscission process. Using the newly released Affymetrix Tomato GeneChip we have identified potential abscission regulatory genes as well as new AZ-specific genes, the expression of which was modified after flower removal. These include: members of the Aux/IAAgene family, ethylene signal transduction-related genes, early and late expressed transcription factors, genes which encode post-translational regulators whose expression was modified specifically in the AZ, and many additional novel AZ-specific genes which were previously not associated with abscission. This microarray analysis allowed us to select an initial set of target genes for further functional analysis by VIGS. Implications: Our success in achieving the two objectives of this feasibility study provides us with a solid basis for further research outlined in the original proposal. This will significantly increase the probability of success of a full 3-year project. Additionally, our feasibility study yielded highly innovative results, as they represent the first direct demonstration of the functional involvement of a TAPG in abscission, and the first microarray analysis of the abscission process. Using these approaches we could identify a large number of genes involved in abscission regulation, initiation and execution, and in auxin-ethylene cross-talk, which are of great importance, and could enable their potential functional analysis by VIGS.