A PROTEOMIC APPROACH TO IDENTIFY PROTEIN INTERACTORS RESPONSIBLE OF FLOWERING TIME DETERMINATION IN THE MODEL SPECIES ARABIDOPSIS THALIANA

Reproductive success in plants is dependent on the timing of the switch from vegetative to reproductive phase coinciding with optimal environmental and developmental conditions. This is a key step that dramatically influences plant productivity, one of the most important aspects in agriculture. Plants have evolved an elaborate regulatory network that integrates endogenous and environmental signals to ensure that flowering occurs when conditions are most favorable. During the last two decades, functional genomics studies have revealed the existence of a complex network of genetic interactions responsible of integrating the different types of signals, both internal and external, that plants receive and that define when plants can enter into the reproductive phase (Liu et al. 2009). However, the molecular characterization of the floral transition process is far from being completed, therefore new studies flanked by new research methods are needed to identify and characterize the proteins and protein complexes that play a key role in the transition to the reproductive phase (Jang et al. 2009). One of these proteins involved in floral transition is SVP (Short Vegetative Phase), a MADS-Box transcription factor studied for a long time at genetic level. It has been reported (Liu et al. 2007) that the ectopic expression of this transcription factor causes a late flowering phenotype in transgenic plants. Moreover, this line develops abnormal leaves and also the flower structure is altered. It is known that many, if not all transcription factors, play their biological role as part of multi-subunit protein complexes, on the other side knowledge on those protein complexes is scarce. In the case of SVP, some interacting partners have been identified via yeast-2-hybrid assays (Gregis et al. 2009), however in planta evidences of such interactions have not been provided, yet. During the last two years, in our laboratory we developed a protocol that enable us to coimmunoprecipitate protein complexes using an antibody against GFP fused to SVP, in order to identify the putative partners that interact together, with SVP, in controlling the flowering time. The analysis of the first data generated a large number of putative candidates involved in acetylation, deacetylation and methylation of histones (Cohen et al. 2009; Berr et al. 2010). A major conclusion that can be drawn from our findings, in agreement with recent publications in this field, is that SVP controls flowering time in Arabidopsis by a chromatin-dependent expression regulation of genes involved in the process. The aim of this project was the validation of the results, by using different methods, and the characterization of these putative partners in order to identify the right composition of different complexes in which SVP interacts with, and the mechanisms behind the regulation of flowering time. The flowering time analysis of mutants of the two most promising candidates GCN5 (GENERAL CONTROL NON-REPRESSIBLE 5) and SDG2 (SET domain protein 2) revealed, in agreement with already published data, their involvement in this mechanism (Bertrand et al. 2003; Guo et al. 2010; Berr et al. 2010). Moreover, preliminary data obtained by analyzing the 35S::SVP-GFP svp/svp SDG2/sdg2 plants, indicate that the floral defect caused by the overexpression of SVP, needs also the presence of SDG2 protein.