Small ubiquitin-like modifier conjugating enzyme with active site mutation acts as dominant negative inhibitor of SUMO conjugation in arabidopsis(F).

Small ubiquitin-like modifier (SUMO) conjugation affects a broad range of processes in plants, including growth, flower initiation, pathogen defense, and responses to abiotic stress. Here, we investigate in vivo and in vitro a SUMO conjugating enzyme with a Cys to Ser change in the active site, and show that it has a dominant negative effect. In planta expression significantly perturbs normal development, leading to growth retardation, early flowering and gene expression changes. We suggest that the mutant protein can serve as a probe to investigate sumoylation, also in plants for which poor genetic infrastructure precludes analysis via loss-of-function mutants.

Distinct roles for Arabidopsis SUMO protease ESD4 and its closest homolog ELS1.

SUMO conjugation affects a broad range of processes in Arabidopsis thaliana, including flower initiation, pathogen defense, and responses to cold, drought and salt stress. We investigated two sequence-related SUMO-specific proteases that are both widely expressed and show that they differ significantly in their properties. The closest homolog of SUMO protease ESD4, ESD4-LIKE SUMO PROTEASE 1 (ELS1, alternatively called AtULP1a) has SUMO-specific proteolytic activity, but is functionally distinct from ESD4, as shown by intracellular localization, mutant phenotype and heterologous expression in yeast mutants. Furthermore, we show that the growth defects caused by loss of ESD4 function are not due to increased synthesis of the stress signal salicylic acid, as was previously shown for a SUMO ligase, indicating that impairment of the SUMO system affects plant growth in different ways. Our results demonstrate that two A. thaliana SUMO proteases showing close sequence similarity have distinct in vivo functions.

Substrates related to chromatin and to RNA-dependent processes are modified by Arabidopsis SUMO isoforms that differ in a conserved residue with influence on desumoylation.

The higher plant Arabidopsis (Arabidopsis thaliana) has eight genes potentially coding for small ubiquitin-related modifier (SUMO) proteins. However, two well-expressed isoforms differ from fungal and animal consensus in a conserved glutamine (Gln) residue situated four residues from the carboxyl terminus. We tested deviations in this position in the background of SUMO1, the isoform with the highest expression level, and found that changes do not prevent conjugation to substrate proteins in vivo. Replacement of this conserved Gln by alanine resulted in a protein that was less readily removed from a substrate by SUMO protease EARLY IN SHORT DAYS4 in an in vitro reaction and apparently led to higher levels of SUMO conjugates when expressed in vivo. We used the SUMO1 variant with the Gln-to-alanine substitution, as well as SUMO3 and SUMO5 (which carry methionine and leucine, respectively, at this position), to enrich in vivo substrates. Identification of the most abundant proteins contained in these fractions indicated that they are involved in DNA-related, or in RNA-dependent, processes, such as regulation of chromatin structure, splicing, or translation. The majority of the identified bona fide substrates contain predicted sumoylation sites. A subset of the proteins was expressed in Escherichia coli and could be sumoylated in vitro.

SUMO conjugation in plants.

Covalent attachment of small proteins to substrates can regulate protein activity in eukaryotes. SUMO, the small ubiquitin-related modifier, can be covalently linked to a broad spectrum of substrates. An understanding of SUMO's role in plant biology is still in its infancy. In this review, we briefly summarize the enzymology of SUMO conjugation (sumoylation), and the current knowledge of SUMO modification in Arabidopsis thaliana (L.) Heynh. and other plants, in comparison to animals and fungi. Furthermore, we assemble a list of potential pathway components in the genome of A. thaliana that have either been functionally defined, or are suggested by similarity to pathway components from other organisms.