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SUMOylation is a posttranslational modification of proteins that is found in the eukaryotic kingdom, but not in bacteria or archeae. During this process, SUMO, the Small Ubiquitin related modifier protein, is covalently attached onto its targets via an enzymatic cascade. SUMOylation can prevent or induce other modifications of the substrate, can lead to conformational changes and generates or abolishes binding interfaces. SUMOylation can therefore change the localization, activity, interactions or life span of a protein. Although SUMOylation is vital in the model plant Arabidopsis thaliana (Saracco et al., 2007), only little is known about regulation and substrates of SUMO conjugation, as the temporary nature of this modification and the fact that only a small subset of substrate is modified at a given time, makes the study of SUMOylation in plants extremely difficult. In this work, several aspects of Arabidopsis SUMOylation are discussed: � An in vitro SUMOylation assay that utilizes plant recombinant proteins was developed. This system allows quick analysis of potential SUMOylation enzymes and substrates in vitro. � The features of a SUMO1 variant, SUMO1 Q90A, in which a conserved glutamine residue at position -4 from the carboxyl terminus is changed to alanine, were analyzed in vitro. It was shown that this mutant variant leads to increased conjugate stability towards Early in Short Days 4 (ESD4), a major SUMO protease of Arabidopsis. As SUMO1 Q90A did not differ during conjugation from the wild type SUMO1 in vitro, this variant might be a valuable tool for future experiments to generate SUMOylated proteins, which are easier to detect and to analyze due to increased stability. � Analysis of the potential SUMO ligases PIAS-LIKE1 (PIL1) and PIAS-LIKE2 (PIL2) indicated a slight contribution to bulk SUMO conjugation and only a minor role in flowering time regulation compared to the already well described SUMO ligase SIZ1, strengthening the importance of SIZ1 as major Arabidopsis SUMO ligase. � Plants with mutation in the SUMO protease ESD4 have growth defects with similarity to those of plants mutated in SIZ1. In contrast to siz1 mutants, however, the growth defect of esd4 mutants is not due to altered levels of the stress hormone salicylic acid. Furthermore, studies of the related SUMO protease Early in Short Days-Like1 (EL1) demonstrated that the latter enzyme does not localize to the nucleus if transiently expressed in Nicotiana benthamiana, and plays no obvious role in the regulation of flowering time, as el1 mutants flower at a time similar to wild type. However, an el1 mutation in the background of ecotype Wassilwskija might cause an altered tissue composition in the shoot. � Analysis of the type III effector protein Factor X of the plant pathogen Xanthomonas campestris (in cooperation with Prof. Ulla Bonas and Robert Szczesny, University Halle) indicated no in vitro activity of this protein as SUMO, Rub1 or Ubiquitin protease. The broad variety of aspects discussed in this work emphasizes the importance and complexity of Arabidopsis SUMOylation and indicates that the understanding of this modification in plants can only be achieved by further studies and identification of in vivo SUMO substrates. In future the SUMOylation assay system, developed in this work, and the described SUMO1 Q90A variant might help to accomplish these tasks. |
