Post-translational modification of proteins by SUMO in Arabidopsis thaliana

Among the systems that modify protein structure, the covalent attachment of small ubiquitin like modifier - SUMO protein to its substrates (sumoylation) represents after ubiquitylation, the best studied example of post-translational modification by a protein modifier. SUMO is a peptide of approximately 100 amino acids that modifies proteins of many organisms including yeast, humans and plants. SUMO is covalently linked to other proteins via a set of specific enzymes, namely SUMO-activating enzyme (SAE), SUMO-conjugating enzyme (SCE) and SUMO ligases. These enzymes are homologous to E1 - E2 - E3 cascade that operates in ubiquitylation. SUMO is emerging as a versatile modifier for a large number of proteins in many different pathways and the consequences of this modification seem to be as diverse as its targets. Arabidopsis thaliana has eight full-length genes with significant similarity to animal and fungal SUMO proteins. Phylogenetic analysis clustered the Arabidopsis SUMO proteins into five subfamilies: SUMO1\2, SUMO3, SUMO5, SUMO 4\6 and SUMO 7\8. To identify and characterize SUMO substrates in plants, we developed transgenic Arabidopsis thaliana lines expressing tagged versions of all the SUMO isoforms. Of all these genes expressed in Arabidopsis, four (SUMO1\2, SUMO3 and SUMO5) are highly expressed and form conjugates with substrate proteins in vivo. Following expression of the affinity tagged SUMO1 and subsequent biochemical enrichment, an array of high molecular weight SUMO1 substrates was revealed on a Coomassie stained gel. MALDI-TOF analysis identified 25 novel potential sumoylation targets in Arabidopsis, some of which carried the consensus sumoylation motif. We were able to demonstrate that one of these identified sumoylation substrates (NAF) is an in vitro target for sumoylation. All SUMO isoforms are made as inactive precursors. They mature by a carboxyl-terminal proteolytic cleavage which yields the mature modifier with exposed carboxyl terminus di-glycine motif. In order to test the requirement for these residues in conjugation reactions, we substituted the double glycine residues with alanine-glycine, glycine-alanine and alanine-alanine in a SUMO1 transgene. These individually modified SUMO1 transgenes were expressed in Arabidopsis. Contrary to the expectation, substrates were visidualized on immunoblotting with all the expressed SUMO variants, which demonstrates the extreme flexibility of the plant SUMO conjugation system. Moreover, an accumulation of SUMO substrates was evident from immunoblot experiments with transgenic Arabidopsis plants expressing a SUMO1 (Q93A) mutant. Furthermore, we investigated Arabidopsis plants with decreased capacity to conjugate SUMO to its target substrates. These plants expressed a mutated version of SUMO conjugating enzyme (AtSCE) in which the active site cysteine residue was changed to serine (C94S). Phenotypic characterization of these plants deficient in sumoylation showed stunted morphology and early flowering characteristics both under short and long day light conditions as compared to the wild type counterparts. Immunoblot analysis revealed that these transgenic lines had lower levels of free endogenous SUMO. Generally, the results suggest that similar to other eukaryotic organisms, many proteins in plants also undergo post-translational modification via sumoylation and this process has functional significance for development and cell biology of Arabidopsis thaliana

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

Federated reinforcement learning based task offloading approach for MEC-assisted WBAN-enabled IoMT

The exponential proliferation of wearable medical apparatus and healthcare information within the framework of the Internet of Medical Things (IoMT) introduces supplementary complexities pertaining to the elevated Quality of Service (QoS) of intelligent healthcare in the forthcoming 6G era. Healthcare services and applications need ultra-reliable data transfer and processing with ultra-low latency and energy usage. Wireless Body Area Network (WBAN) and Mobile Edge Computing (MEC) technologies enabled IoMT to handle large amounts of data sensing, transmission, and processing while maintaining good QoS. Traditional frame aggregation (FA) systems in WBAN, on the other hand, create an excessive number of control frames during data transmission, resulting in significant latency and energy consumption, as well as a lack of flexibility. A Federated Reinforcement Learning (FRL) based TO Approach is recommended in this research. In the beginning, different types of service-related information were separated into queues with equal QoS needs. The duration of the FA was then automatically determined by the aggregation vertex based on energy consumption, latency, and throughput using FRL. Finally, based on the existing status, the amount of tasks offloaded was determined. The simulation results demonstrate that, as compared to the baseline schemes, the suggested FRLTO efficiently reduces energy consumption and latency while enhancing throughput and total WBAN utilization. Numerical results show that the proposed scheme improves the throughput by 37.06% and reduced the energy consumption by around 69.84% and time delay by about 6.23%, as compared to the state-of-the-art existing baseline schemes

Substrates Related to Chromatin and to RNA-Dependent Processes Are Modified by Arabidopsis SUMO Isoforms That Differ in a Conserved Residue with Influence on Desumoylation1[W][OA]

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