Reconstitution of Arabidopsis thaliana SUMO Pathways in E. coli: Functional Evaluation of SUMO Machinery Proteins and Mapping of SUMOylation Sites by Mass Spectrometry

Choi

et al. 2011

Molecules and Cells

Reversible conjugation of the small ubiquitin modifier (SUMO) peptide to proteins (SUMOylation) plays important roles in cellular processes in animals and yeasts. However, little is known about plant SUMO targets. To identify SUMO substrates in Arabidopsis and to probe for biological functions of SUMO proteins, we constructed 6xHis-3xFLAG fused AtSUMO1 (HFAtSUMO1) controlled by the CaMV35S promoter for transformation into Arabidopsis Col-0. After heat treatment, an increased sumoylation pattern was detected in the transgenic plants. SUMO1-modified proteins were selected after two-dimensional gel electrophoresis (2-DE) image analysis and identified using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We identified 27 proteins involved in a variety of processes such as nucleic acid metabolism, signaling, metabolism, and including proteins of unknown functions. Binding and sumoylation patterns were confirmed independently. Surprisingly, MCM3 (At5G46280), a DNA replication licensing factor, only interacted with and became sumoylated by AtSUMO1, but not by SUMO1ΔGG or AtSUMO3. The results suggest specific interactions between sumoylation targets and particular sumoylation enzymes.

Section: Molecules and Cellsmentioning

confidence: 99%

Section: Agrobacterium-mediated Transient Expression Assay In Tobaccomentioning

confidence: 99%

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Identification and Molecular Properties of SUMO-Binding Proteins in Arabidopsis

Choi

et al. 2011

Molecules and Cells

“…The At2g20310 cDNA was cloned into the NcoI/XhoI sites of pET28a vector and NotI/XhoI sites of pET28c vector to fuse in frame to the 3' end of the coding sequences of T7-His 6 . This system contains E1 enzyme (50 ng of His 6 -Fub2/Rad31), E2 enzyme (100ng of His 6 -Hus5), E3 enzyme (200ng of His 6 -Pli1), Pmt3-GG (1µg), 40 mM ATP and T7-His 6 -At2g20310 protein substrate (100 ng) in SUMOylation buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 1 mM dithiothreitol, 5 mM ATP) (Okada et al, 2009). The products were separated and detected using anti-T7 monoclonal antibody (Novagen).…”

Section: In Vitro Sumo Conjugation Assaymentioning

confidence: 99%

“…The binding of SUMO to targets may be assisted directly by E2 or E3. Interestingly, E3 was proven to be an unessential requirement for SUMO conjugation to target proteins, displayed by the fact that SUMOylation can occur in reconstituted system (Okada et al, 2009).Although SUMOylation occurs only in a small portion of the total pool of the protein, SUMO has been shown to play important roles in diverse processes such as nucleo-cytoplasmic transporter, chromosome segregation, gene expression, chromatin structure, signal transduction, and genome maintenance (Geiss-Friedlander & Melchior, 2007). Unlike ubiquitination, SUMOylation is not known to target proteins for degradation, but rather is thought to regulate protein-protein interactions, alter the subcellular localization and/or activity of targets, and antagonize ubiquitin-dependent degradations (Martin, Wilkinson, Nishimune, & Henley, 2007;Miller & Vierstra, 2011;Xu & Yang, 2013).…”

Section: Introductionmentioning

confidence: 99%

Characterization of The Interaction Between RIN13 (RPM1-Interacting13) and Sumo Proteins in Arabidopsis Thaliana

et al. 2017

Self Cite

Small Ubiquitin-related MOdifier (SUMO) proteins can be found in many organisms, including A. thaliana, which possesses 9 SUMO genes. SUMO binds to various target proteins in a reversible reaction called SUMOylation. SUMOylation participates in transcription, chromosome organization, proteins localizations and stress responses. Our study showed that RIN13 (RPM1-Interacting13/At2g20310) is a target of SUMOylation, which was initially found by interaction between this protein and AtSCE1a (E2). Recent report showed that overexpression of RIN13 enhanced the resistance to pathogen without inducing hypersensitive response. However, the molecular interaction between RIN13 and SUMO proteins and its significance have not been studied yet. Thus, our study aimed to characterize the Interaction between RIN13 and SUMO proteins in A. thaliana. The result showed an isoform-specific SUMOylation between RIN13 and SUMO proteins. RIN13 is SUMOylated by SUMO1, 2, 3, and 5. Though expressed ubiquitously in A.thaliana, fluorescence microscopy showed that RIN13 localizes subcellularly in the nuclear body. Moreover, complete abolishment of SUMOylation with inactive E2 suggests the exclusion of RIN13 from nuclear body. These results showed that SUMOylation affected RIN13 localization, and indirectly influenced its interaction to other proteins and putative function. This paper presents evidence of RIN13 SUMOylation. Furthermore, RIN13 function in pathogenic resistance is shown to be supported by SUMOylation. Thus, this study enhanced the understanding of SUMO in plants and served as reference to molecular studies concerning post-translational modification of SUMO. How to Cite

Improving Crop Resistance to Abiotic Stress

Make Your Best – MYB Transcription Factors for Improving Abiotic Stress Tolerance in Crops

Pitzschke

2012

Too hot, too dry, too cold, and too bright. Field crops grow and reproduce in a dynamic and unpredictably changing environment. Adverse climate conditions severely impair plant growth and development. In the face of the growing world population and the climate change, humanity cannot take food production for granted. There is an increasing demand for crops with improved stress tolerance and yield. Understanding the molecular mechanisms that mediate stress adaptation and applying this knowledge to engineering stress-resistant crops is therefore a key to ensure world food security.MYB domain-containing proteins form a family of transcription factors involved in a diversity of stress-related responses in plants. Interfering with the activity of individual family members often correlates with altered stress tolerance. MYB transcription factors have been thoroughly studied and functionally characterized not only in the model plant Arabidopsis thaliana but also in other plant species. Heterologous expression approaches and phylogenetic analyses indicate a good degree of functional conservation. This conservation will facilitate knowledge transfer, and thus tomorrows farmers may benefit from todays discoveries in Arabidopsis. This chapter reviews the role of MYB transcription factors in abiotic stress signaling. While selected examples from Arabidopsis are described, emphasis is given to MYB proteins in field crops. The potentials and limitations of using heterologous expression approaches for genetic engineering of crops with improved stress tolerance are discussed. One part is devoted to mechanisms that regulate MYB protein abundance and activity. A list of valuable data resources (transcription factor databases, transcriptome studies) shall provide fast access to detailed information and bioinformatics tools for researchers interested in a particular plant species.