Objective The aim of this project is to describe proteolytic mechanisms that regulate cell cycle during organ growth in plants. For this purpose we propose to functionally characterize two RING finger proteins for which preliminary data suggest impact on cell cycle regulation in Arabidopsis thaliana. Our first major objective (1) is to determine through which proteins the RING proteins mediate cell cycle regulation by screening for interacting proteins, and by testing their functional interaction. The second major ob jective (2) is to determine how the RING proteins mediate regulation of plant growth by analyzing their regulation and function in plants. 1) For the first objective E3 ligase activity of the RING proteins will be determined by in vitro ubiquitination. Yea st-2-Hybrid and Tandem Affinity Purification (TAP) techniques will be used to screen for RING interacting proteins. The true substrates will be confirmed by determining their functional interaction with the RING proteins in pull down and ubiquitination ass ays in vitro and in plant. 2) The second objective will be addressed in cell cultures and by developmental assays. Levels of endogenous and tagged RING proteins will be analyzed during cell cycle and in plant. RING protein function in plant will be determi ned by overexpression and mutation effects of the RING proteins on growth parameters such as; organ growth, biomass, yield, and on kinematic parameters of growth rate and cell cycle duration. According to our working model these two RING proteins act as ¿ positive regulators¿ of cell cycle by mediating degradation of ¿negative regulators¿ of cell cycle proteins. As a result when the RING function is impaired in the Ring mutants the degradation of negative regulator is suppressed resulting in repression o f cell cycle activity. The multidisciplinary approach applying proteomic analysis, functional assays and phenotypic characterization of transgenic plants will allow us to comprehensively test our working model. Fields of science natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsnatural sciencesbiological sciencesgeneticsmutationagricultural sciencesagricultural biotechnologybiomass Keywords RING finger protein cell cycle organ growth protein degradation ubiquitination Programme(s) FP6-MOBILITY - Human resources and Mobility in the specific programme for research, technological development and demonstration "Structuring the European Research Area" under the Sixth Framework Programme 2002-2006 Topic(s) MOBILITY-2.1 - Marie Curie Intra-European Fellowships (EIF) Call for proposal FP6-2002-MOBILITY-5 See other projects for this call Funding Scheme EIF - Marie Curie actions-Intra-European Fellowships Coordinator FLANDERS INTERUNIVERSITY INSTITUTE FOR BIOTECHNOLOGY EU contribution No data Address Belgium See on map Total cost No data
