The ubiquitin system and jasmonate signaling

The ubiquitin (Ub) system is involved in most, if not all, biological processes in eukaryotes. The major specificity determinants of this system are the E3 ligases, which bind and ubiquitinate specific sets of proteins and are thereby responsible for target recruitment to the proteasome or other cellular processing machineries. The Ub system contributes to the regulation of the production, perception and signal transduction of plant hormones. Jasmonic acid (JA) and its derivatives, known as jasmonates (JAs), act as signaling compounds regulating plant development and plant responses to various biotic and abiotic stress conditions. We provide here an overview of the current understanding of the Ub system involved in JA signaling

Design and development of a decision aid to enhance shared decision making by patients with an asymptomatic abdominal aortic aneurysm

Dirk T Ubbink1,2, Anouk M Knops1, Sjaak Molenaar1, Astrid Goossens11Department of Quality Assurance and Process Innovation and 2Department of Surgery, Academic Medical Center, Amsterdam, The NetherlandsObjective: To design, develop, and evaluate an evidence-based decision aid (DA) for patients with an asymptomatic abdominal aortic aneurysm (AAA) to inform them about the pros and cons of their treatment options (ie, surgery or watchful observation) and to help them make a shared decision.Methods: A multidisciplinary team defined criteria for the desired DA as to design, medical content and functionality, particularly for elderly users. Development was according to the international standard (IPDAS). Fifteen patients with an AAA, who were either treated or not yet treated, evaluated the tool.Results: A DA was developed to offer information about the disease, the risks and benefits of surgical treatment and watchful observation, and the individual possibilities and threats based on the patient’s aneurysm diameter and risk profile. The DA was improved and judged favorably by physicians and patients.Conclusion: This evidence-based DA for AAA patients, developed according to IPDAS criteria, is likely to be a simple, user-friendly tool to offer patients evidence-based information about the pros and cons of treatment options for AAA, to improve patients’ understanding of the disease and treatment options, and may support decision making based on individual values.Keywords: decision support techniques, research design, program development, abdominal aortic aneurysm, decision makin

A multiSite gateway™ vector set for the functional analysis of genes in the model Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Recombinatorial cloning using the Gateway^TM technology has been the method of choice for high-throughput omics projects, resulting in the availability of entire ORFeomes in Gateway^TM compatible vectors. The MultiSite Gateway^TM system allows combining multiple genetic fragments such as promoter, ORF and epitope tag in one single reaction. To date, this technology has not been accessible in the yeast <it>Saccharomyces cerevisiae</it>, one of the most widely used experimental systems in molecular biology, due to the lack of appropriate destination vectors.</p> <p>Results</p> <p>Here, we present a set of three-fragment MultiSite Gateway^TM destination vectors that have been developed for gene expression in <it>S. cerevisiae</it> and that allow the assembly of any promoter, open reading frame, epitope tag arrangement in combination with any of four auxotrophic markers and three distinct replication mechanisms. As an example of its applicability, we used yeast three-hybrid to provide evidence for the assembly of a ternary complex of plant proteins involved in jasmonate signalling and consisting of the JAZ, NINJA and TOPLESS proteins.</p> <p>Conclusion</p> <p>Our vectors make MultiSite Gateway^TM cloning accessible in <it>S. cerevisiae</it> and implement a fast and versatile cloning method for the high-throughput functional analysis of (heterologous) proteins in one of the most widely used model organisms for molecular biology research.</p

Heat adaptation of Escherichia coli K12: Effect of acid and glucose

AbstractThe objective of this work is to investigate the effect of the (possible) acid adaptation during growth in a glucose rich environment on the heat resistance of Escherichia coli K12 MG1655. E. coli cells were grown in TSB and/or TSB dextrose free broth until they reached the stationary phase. Afterwards, the stationary phase cells were added in TSB and/or TSB dextrose free broth and inactivation took place at 54oC and 58oC. It was observed that growth in a glucose rich environment leads to an increased heat resistance, most likely due to a certain level of acid and further heat adaptation via cross protection

Glutaredoxin GRXS17 associates with the cytosolic iron-sulfur cluster assembly pathway

Cytosolic monothiol glutaredoxins (GRXs) are required in iron-sulfur (Fe-S) cluster delivery and iron sensing in yeast and mammals. In plants, it is unclear whether they have similar functions. Arabidopsis (Arabidopsis thaliana) has a sole class II cytosolic monothiol GRX encoded by GRXS17. Here, we used tandem affinity purification to establish that Arabidopsis GRXS17 associates with most known cytosolic Fe-S assembly (CIA) components. Similar to mutant plants with defective CIA components, grxs17 loss-of-function mutants showed some degree of hypersensitivity to DNA damage and elevated expression of DNA damage marker genes. We also found that several putative Fe-S client proteins directly bind to GRXS17, such as XANTHINE DEHYDROGENASE1 (XDH1), involved in the purine salvage pathway, and CYTOSOLIC THIOURIDYLASE SUBUNIT1 and CYTOSOLIC THIOURIDYLASE SUBUNIT2, both essential for the 2-thiolation step of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification of tRNAs. Correspondingly, profiling of the grxs17-1 mutant pointed to a perturbed flux through the purine degradation pathway and revealed that it phenocopied mutants in the elongator subunit ELO3, essential for the mcm5 tRNA modification step, although we did not find XDH1 activity or tRNA thiolation to be markedly reduced in the grxs17-1 mutant. Taken together, our data suggest that plant cytosolic monothiol GRXs associate with the CIA complex, as in other eukaryotes, and contribute to, but are not essential for, the correct functioning of client Fe-S proteins in unchallenged conditions

The RING E3 ligase KEEP ON GOING modulates JASMONATE ZIM-DOMAIN12 stability

Jasmonate (JA) signaling in plants is mediated by the JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the activity of several transcription factors regulating JA-inducible gene expression. The hormone JA-isoleucine triggers the interaction of JAZ repressor proteins with the F-box protein CORONATINE INSENSITIVE1 (COI1), part of an S-phase kinase-associated protein1/Cullin1/F-box protein COI1 (SCFCOI1) E3 ubiquitin ligase complex, and their degradation by the 26S proteasome. In Arabidopsis (Arabidopsis thaliana), the JAZ family consists of 13 members. The level of redundancy or specificity among these members is currently not well understood. Here, we characterized JAZ12, encoded by a highly expressed JAZ gene. JAZ12 interacted with the transcription factors MYC2, MYC3, and MYC4 in vivo and repressed MYC2 activity. Using tandem affinity purification, we found JAZ12 to interact with SCFCOI1 components, matching with observed in vivo ubiquitination and with rapid degradation after treatment with JA. In contrast to the other JAZ proteins, JAZ12 also interacted directly with the E3 RING ligase KEEP ON GOING (KEG), a known repressor of the ABSCISIC ACID INSENSITIVE5 transcription factor in abscisic acid signaling. To study the functional role of this interaction, we circumvented the lethality of keg loss-of-function mutants by silencing KEG using an artificial microRNA approach. Abscisic acid treatment promoted JAZ12 degradation, and KEG knockdown led to a decrease in JAZ12 protein levels. Correspondingly, KEG overexpression was capable of partially inhibiting COI1-mediated JAZ12 degradation. Our results provide additional evidence for KEG as an important factor in plant hormone signaling and a positive regulator of JAZ12 stability

Із зали засідань Президії НАН України

20 червня 2012 року відбулося виїзне засідання Президії Національної академії наук України на запрошення президента — генерального конструктора Державного підприємства «АНТОНОВ» академіка НАН України Д.С. Ківи

FRS7 and FRS12 recruit NINJA to regulate expression of glucosinolate biosynthesis genes

The sessile lifestyle of plants requires accurate physiology adjustments to be able to thrive in a changing environment. Plants integrate environmental timing signals to control developmental and stress responses. Here, we identified Far1 Related Sequence (FRS) 7 and FRS12, two transcriptional repressors that accumulate in short-day conditions, as regulators of Arabidopsis glucosinolate (GSL) biosynthesis. Loss of function of FRS7 and FRS12 results in plants with increased amplitudes of diurnal expression of GSL pathway genes. Protein interaction analyses revealed that FRS7 and FRS12 recruit the NOVEL INTERACTOR OF JAZ (NINJA) to assemble a transcriptional repressor complex. Genetic and molecular evidence demonstrated that FRS7, FRS12 and NINJA jointly regulate the expression of GSL biosynthetic genes, and thus constitute a molecular mechanism that modulates specialized metabolite accumulation