Ubiquitin and cell death in plants

Modification of proteins by covalent attachment of the small conserved protein ubiquitin, called ubiquitylation, is central to many regulatory processes in plants and in animals. We are specifically interested in the contribution of this process to plant cell death programs. The focus of the application lies on two groups of ubiquitylation enzymes that were linked by previous work to cell death processes in plants. However, neither the exact molecular activity, nor the physiological role of these ubiquitylation components is currently well understood. Similar to other components associated with cell death, the proteins chosen for these studies presumably have additional roles in other processes, for instance in stress response or in development. The application puts most emphasis on the molecular characterization of the proteins of interest. The additional knowledge stemming from these studies shall help to understand their contribution to cell death and to other processes. One of the two ubiquitylation reactions chosen for detailed studies employs ubiquitin conjugation enzymes that are unusually large, and are distantly related to a mammalian protein with anti-apoptotic activity. The intracellular location, interaction partners and the in vitro activity of these proteins shall be investigated. The second type of ubiquitylation reaction to be investigated is called N-end rule pathway. Investigations include a search for novel enzymes of this pathway and the characterization of substrates of the pathway that are relevant to a plant-specific form of cell death called senescence. Methods of investigation include in vitro studies of recombinant proteins, yeast two hybrid analysis, in vivo expression and microscopic observation of proteins fused to fluorescent marker proteins, elucidation of substrate proteins by specific labeling techniques and mass spectroscopic analysis, and physiological experiments with mutants impaired in the function of the investigated components. The work shall deepen our understanding of plant cell death and, more generally, of regulatory processes in plants, and thereby contribute to improvements in agriculture.

Modification of proteins after their synthesis, by linking them covalently to so-called modifier proteins, was first described some 20 years ago, and several different modifier proteins have been discovered since. In a cell, protein modification often connects internal or external signals with the appropriate response, be it a change in enzyme activity, or alterations in gene transcription. In our work, the two modifiers ubiquitin and SUMO (small ubiquitin-related modifier) of the model plant Arabidopsis thaliana are in the focus of interest. FWF grant P21215-B12, Ubiquitin and cell death in plants, supported studies on these two modifiers and their roles in cell death programs and in response to unfavorable environmental conditions. Previously uncharacterized components of both pathways were studied by biochemical analysis and by generation of mutants in the respective functions. The mutants were subjected to detailed analysis, to find physiological responses that depend on these components.In particular, mutants in all components of a ubiquitin conjugation route called N-end rule pathway were generated and studied. In collaboration with other laboratories, these mutants were found to be less proficient in their reaction to environmental perturbations. Another group of enzymes that mediates ubiquitin conjugation was found to participate in responses to reactive oxygen species, which are common alarm signals of plants. We had previously described a role for ubiquitin in cell death, manifested after inhibiting ubiquitin conjugation in A. thaliana with the help of a modified ubiquitin protein, which acts as a dominant inhibitor. The nature of the cell death program initiated after inhibitor expression was studied in more detail. The second modifier under study, SUMO, is known for multiple contributions to plant survival following environmental stress. We characterized two sequence-related enzymes that are activated if plants grow on salty soils, or under drought stress. It is a particularly rewarding task of molecular plant sciences to investigate the mechanisms that help plants to survive stressful environmental conditions. The genes studied by us in Arabidopsis thaliana are conserved in crops, and it is clear that elucidation of stress resistance pathways in the model plant will help to cope with environmental changes, which are the expected challenges to future agriculture.