Sumoylation and Phosphorylation in Plant Stress Responses

As sessile organisms, it is of particular importance for plants to cope with stressful environmental conditions. In this application, we focus on two protein modifications that both contribute to plant survival under adverse conditions. One type of modification is an enzyme cascade that links the small modifier protein SUMO to substrates. This modification has a crucial role in plant responses to heat, cold, drought and other stresses. Many of its features, however, remain to be characterized. The other modification, addition of phosphate residues to proteins, is extensively analyzed, but due to its complexity, its contributions to plant stress response is also incompletely understood. Recently published data suggest that these two modifications are coordinated in vivo, to achieve optimal protection of plant life. We want to analyze this cross-talk in a systematic way, in a collaborative effort between one lab with expertise in small protein modifiers, and one lab with expertise in phospho-proteomics. We plan to use biochemical, genetic and cell biology methods to characterize two novel components of the SUMO conjugation system of Arabidopsis. Mutants in these and in previously characterized components will be used to investigate their phospho-proteome. Comparison with wild type plants shall uncover compensatory changes in phosphorylation, and regulatory cross-talk between sumoylation and phosphorylation via critical substrates. Candidate components and substrates of the SUMO-phospho cross-talk shall be studied in the second phase of the project, to uncover the underlying regulatory circuitry.

The FWF financed project Protein Sumoylation and Phosphorylation in Plant Stress Responses resulted in significant insights into how plant cells cope with adverse environmental influences, and how they coordinate two different ways of changing protein properties, namely the attachment of the small ubiquitin-related modifier protein SUMO (sumoylation), and of phosphate residues (phosphorylation) to the protein substrates. A novel enzyme activity was characterized that exists in higher, but apparently not in single-celled organisms. The characterized enzyme forms chains of SUMO. Critical properties of this enzyme were elucidated. It was shown that SUMO chains regulate responses to salt and drought stress. We postulated that the attachment of such chains to a substrate protein results in its degradation via the ubiquitin-proteasome pathway. In the second part of the project, the relationship of protein modification by phosphorylation and of protein modification by attachment of SUMO was investigated. To this end, the entire set of proteins (the proteome) and of phosphorylated proteins was investigated in plants with mutations in the sumoylation process. We found that sumoylation and phosphorylation have a subset of common targets. Both modifications help plants to cope with adverse environmental influences. We therefore concluded that critical substrates for both modifications are important regulators of stress responses, and that both modifications impact on optimizing growth and survival of plants, in part by influencing the same response pathways. The proteome analysis also resulted in the identification of candidate substrates for the postulated SUMO chain attachment degradation pathway. Proteins were identified that have increased abundance in plants with defect in SUMO chain formation, even though the synthesis rate of these proteins is unchanged. These candiates can be further investigated in future projects.Because all investigated regulation processes exist not only in the model plant Arabidopsis thaliana, but also in crop plants, the projects results help to improve food supply under difficult environmental conditions.