ISIDE

The gametophytic self-incompatibility (GSI) affects production in agricultural species of great economic interest such as clementine and other citrus (Rutaceae), apple and pear (Rosaceae; Pyrinae). ISIDE will explore the mechanisms controlling GSI in clementine and Pyrinae species by using integrated genomic, transcriptomic, metabolomic, proteomic and cytohistological approaches. In the Pyrinae, GSI acts through a pollen-pistil recognition mechanism, which is controlled by a single S-locus. The S-locus contains a gene encoding the S-RNase, the female pistil determinant, and several genes encoding F-box proteins (SFBB) that collaboratively act as male (pollen) determinants. Based on the current recognition model, a single SFBB is supposed to bind, specifically, one or a few S-RNases, but this mechanism is still largely unexplored. To investigate the SFBB specificity of action, selected apple and pear S-haplotypes will be used for characterizing the intra- and inter-haplotypic SFBB variants, as a prerequisite for the subsequent analyses of the binding properties of S-RNases with different SFBB proteins. Conversely, molecular mechanisms of clementine GSI are still elusive although some key genes have been identified, among which an F-box gene and three genes encoding aspartic acid-rich proteins, involved in calcium sequestration. Overexpression of these genes in the heterologous system Nicotiana will uncover their biological function and more details will be achieved by biochemical assays. The analysis of metabolic profile in pollen, style and pollinated styles with compatible and self-incompatible pollen will complement the gene expression analyses. In incompatible crosses, GSI triggers several cellular responses, which culminate with programmed cell death (PCD). These events will be explored by analysing the interactions between S-RNase, cytoskeleton-associated proteins and transglutaminase (TGase), that has actin and tubulin as substrates. The cytoskeleton promotes organelle movement and the deposition of the pollen tube cell wall, therefore the effects of the response induced by recombinant S-RNase on the cell wall structure will be studied thereby evaluating the quantitative/qualitative variation in the main components, and the mechanism of internalization and degradation of S-RNase. PCD induction in pollen will be investigated by treatments with incompatible S-RNases. Associated variations in the levels of reactive oxygen species (ROS), cytosolic calcium and cytoskeleton will be explored. Cyto-histological and biochemical analyses will help to identify mechanisms of action and mediators, shared or not-shared by the species under study, to integrate genomic, transcriptomic and proteomic results. ISIDE will shed light on reproductive biology in fruit tree species, allowing prediction of self-compatibility or incompatibility of new varieties, of cross-compatibility between cultivars and contributing to sustainable orchard management.