Functional characterization of an E3 ubiquitin ligase involved in plant response to abiotic stress

Protein ubiquitination is a post-translational modification that targets protein substrates for 26S proteasome-mediated degradation. It is based on the covalent attachment of the 76-amino acid eukaryotic molecule, ubiquitin, to substrate proteins. Protein ubiquitination plays a key role in a wide variety of cellular processes such as hormone signaling, DNA repair, biotic and abiotic stress response, cell cycle regulation. Ubiquitin conjugation is a multistep reaction, sequentially involving three enzymes referred to as E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase). In Arabidopsis thaliana more than one thousand of genes code for E3 ubiquitin enzymes that specifically recognize target proteins. In a previous work we isolated an E3 ubiquitin ligase early induced during cold/light stress in durum wheat. In this work several approaches were initiated to functional characterize the E3. To test its functionality in vitro an ubiquitination assay was carried out and the subcellular localization was determined. The involvement of the E3 in drought stress was also investigated. Moreover a wheat cDNA library from cold treated leaves of Triticum durum has been produced and screened by two-hybrid system to isolate potential E3 interactors and ubiquitination targets. In parallel was developed a system to identify E3 targets on large scale in Arabidopsis. Preliminary results of this work will be shown.