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 Â¿ positive regulatorsÂ¿ of cell cycle by mediating degradation of Â¿negative regulatorsÂ¿ 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.
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