Protein modification by SUMO (small ubiquitin-related modifier) has emerged as an essential regulatory mechanism in eukaryotes. Even though the molecular mechanisms of SUMO conjugation/deconjugation are conserved, the number of SUMO machinery components and their degree of conservation are specific to each organism. In the present paper, we show data contributing to the notion that the four expressed Arabidopsis SUMO paralogues, AtSUMO1, 2, 3 and 5, have functionally diverged to a higher extent than their human orthologues. We have explored the degree of conservation of these paralogues and found that the surfaces involved in E1-activating enzyme recognition, and E2-conjugating enzyme and SIM (SUMO-interacting motif) non-covalent interactions are well conserved in AtSUMO1/2 isoforms, whereas AtSUMO3 shows a lower degree of conservation, and AtSUMO5 is the most divergent isoform. These differences are functionally relevant, since AtSUMO3 and 5 are deficient in establishing E2 non-covalent interactions, which has not been reported for any naturally occurring SUMO orthologue. In addition, AtSUMO3 is less efficiently conjugated than AtSUMO1/2, and AtSUMO5 shows the lowest conjugation level. A mutagenesis analysis revealed that decreases in conjugation rate and thioester-bond formation are the result of the non-conserved residues involved in E1-activating enzyme recognition that are present in AtSUMO3 and 5. The results of the present study support a role for the E1-activating enzyme in SUMO paralogue discrimination, providing a new mechanism to favour conjugation of the essential AtSUMO1/2 paralogues.

Sumoylation is an essential posttranslational modification that participates in many biological processes including stress responses. However, little is known about the mechanisms that control Small Ubiquitin-like MOdifier (SUMO) conjugation in vivo. We have evaluated the regulatory role of the heterodimeric E1 activating enzyme, which catalyzes the first step in SUMO conjugation. We have established that the E1 large SAE2 and small SAE1 subunits are encoded by one and three genes, respectively, in the Arabidopsis genome. The three paralogs genes SAE1a, SAE1b1, and SAE1b2 are the result of two independent duplication events. Since SAE1b1 and SAE1b2 correspond to two identical copies, only two E1 small subunit isoforms are present in vivo: SAE1a and SAE1b. The E1 heterodimer nuclear localization is modulated by the C-terminal tail of the SAE2 subunit. In vitro, SUMO conjugation rate is dependent on the SAE1 isoform contained in the E1 holoenzyme and our results suggest that downstream steps to SUMO-E1 thioester bond formation are affected. In vivo, SAE1a isoform deletion in T-DNA insertion mutant plants conferred sumoylation defects upon abiotic stress, consistent with a sumoylation defective phenotype. Our results support previous data pointing to a regulatory role of the E1 activating enzyme during SUMO conjugation and provide a novel mechanism to control sumoylation in vivo by diversification of the E1 small subunit.

Protein modification by SUMO modulates essential biological processes in eukaryotes. SUMOylation is facilitated by sequential action of the E1-activating, E2-conjugating, and E3-ligase enzymes. In plants, SUMO regulates plant development and stress responses, which are key determinants in agricultural productivity. To generate additional tools for advancing our knowledge about the SUMO biology, we have developed a strategy for inhibiting in vivo SUMO conjugation based on disruption of SUMO E1-E2 interactions through expression of E1 SAE2 domain. Targeted mutagenesis and phylogenetic analyses revealed that this inhibition involves a short motif in SAE2 highly divergent across kingdoms. Transgenic plants expressing the SAE2 domain displayed dose-dependent inhibition of SUMO conjugation, and have revealed the existence of a post-transcriptional mechanism that regulates SUMO E2 conjugating enzyme levels. Interestingly, these transgenic plants displayed increased susceptibility to necrotrophic fungal infections by Botrytis cinerea and Plectosphaerella cucumerina. Early after fungal inoculation, host SUMO conjugation was post-transcriptionally downregulated, suggesting that targeting SUMOylation machinery could constitute a novel mechanism for fungal pathogenicity. These findings support the role of SUMOylation as a mechanism involved in plant protection from environmental stresses. In addition, the strategy for inhibiting SUMO conjugation in vivo described in this study might be applicable in important crop plants and other non-plant organisms regardless of their genetic complexity.

Post-translational modification by Small Ubiquitin-related Modifier (SUMO) is an essential regulatory mechanism in eukaryotes. In the cell, SUMO conjugates are highly enriched in the nucleus and, consistently, SUMOylation machinery components are mainly nuclear. Nonetheless, cytosolic SUMO targets also exist and the mechanisms that facilitate SUMO conjugation in the cytosol are unknown. Here, we show that the nuclear localization of the Arabidopsis SUMO activating enzyme large subunit SAE2 is dependent on two nuclear localization signals, the canonical NLS1 and the non-canonical NLS2 identified and validated here. NLS2 is proteolytic processed from SAE2 during seed development, facilitating SAE2 enrichment in the cytosol. Results obtained using transgenic plants expressing different SAE2 proteoforms suggest that SAE2 cytosolic enrichment could constitute a rapid signal for growth arrest. Phylogenetic studies indicated that the Arabidopsis NLS1-NLS2 structural organization is conserved only in seed plants, providing a potential evolutionary role of cytosolic SUMOylation in seed appearance.

Plants display a high diversification degree of the SUMO conjugation machinery, which could confer a biological specialization of the different isoforms. For instance, the two essential Arabidopsis SUMO isoforms, SUMO1/2, display the highest conjugation rate when compared to SUMO3 and 5, suggesting that their specific biochemical properties may be linked to their biological specialization. In order to study the biochemical properties of plant SUMO conjugation systems, quantitative biochemical assays must be performed. We will present a detailed protocol for reconstituting an in vitro SUMO conjugation assay covering all steps from protein preparation to assay development.

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