Studies on the functional characterization of specifier proteins involved in glucosinolate metabolism of the Brassicaceae

Pflanzen der Brassicales nutzen das Glucosinolat-Myrosinase-System zur Verteidigung gegen Fraßfeinde und Pathogene. Bei Gewebeverletzung werden Glucosinolate durch Myrosinasen unter Bildung giftiger Isothiocyanate hydrolysiert. In Anwesenheit spezifizierender Proteine entstehen alternative Produkte. Nitril-spezifizierende Proteine (NSPs) führen zur Bildung von Nitrilen. Arabidopsis thaliana besitzt fünf NSP-Gene (AtNSP1-AtNSP5). Im Rahmen dieser Arbeit wurde der Einfluss von AtNSP1-AtNSP5 auf die Bildung der Glucosinolat-Abbauprodukte in Homogenaten der Samen und Keimlinge von A. thaliana Col-0 untersucht. Um die Funktion spezifizierender Proteine besser zu verstehen, sollte ihre subzelluläre Lokalisation geklärt werden. Außerdem sollte versucht werden, ihre Interaktion mit der Myrosinase TGG1 in planta nachzuweisen. In Homogenaten der Samen und Keimlinge von A. thaliana Col-0 wurden Glucosinolate vorherrschend zu Nitrilen abgebaut. Die Analyse der T-DNA-Mutanten nsp2-1 und nsp2-2 zeigte, dass AtNSP2 in Samen für die Nitrilbildung verantwortlich ist. Im Homogenat von Keimlingen der nsp1-1-Mutante war die Nitrilbildung gegenüber dem Wildtyp stark reduziert. Daher scheint AtNSP1 in Keimlingen verantwortlich für die Nitrilbildung zu sein. Zudem könnten AtNSP3, AtNSP4 und AtNSP5 zur Nitrilbildung in Keimlingen beitragen. Zur Analyse der subzellulären Lokalisation wurden AtNSP1, AtNSP2, AtNSP4, AtNSP5, At3g07720 und das Thiocyanat-formende Protein aus Thlaspi arvense in Fusion mit dem Fluoreszenzprotein Venus transient in Nicotiana benthamiana exprimiert. Alle untersuchten Proteine waren im Cytosol lokalisiert. Zur Untersuchung der Interaktion zwischen TGG1 und AtNSP3 in planta mittels bimolekularer Fluoreszenz-Komplementation wurden die in verschiedenen Kompartimenten von N. benthamiana-Zellen in Fusion mit Reporter-Fragmenten produzierten Proteine in einem Blattextrakt untersucht. Der Nachweis scheiterte bei Verwendung der Reporter-Fragmente VYNE und SCYCE an einer starken Hintergrundfluoreszenz. Mit mCherry als Reporter war die Hintergrundfluoreszenz geringer, aber kein Fluoreszenzmaximum im Interaktionsansatz nachweisbar. Bei cytosolischer Expression beider Interaktionspartner konnte nur die unglykosylierte Myrosinase (TGG1-oSP) betrachtet werden. Eine Interaktion zwischen TGG1-oSP und AtNSP3 ließ sich dann nachweisen. Entsprechende Untersuchungen mit TGG1-oSP und AtNSP5 bzw. At3g07720 führten nicht zum Nachweis einer Interaktion.The glucosinolate-myrosinase-system is an important defence mechanism against herbivores and pathogens in members of the Brassicales. If tissue is disrupted, myrosinases hydrolyze glucosinolates and toxic isothiocyanates are formed. In presence of specifier proteins, alternative hydrolysis products are generated. The presence of nitrile-specifier proteins (NSPs) leads to the formation of nitriles. Arabidopsis thaliana possesses five NSP genes (AtNSP1-AtNSP5). The aim of the present work was to analyse the role of AtNSP1-AtNSP5 in nitrile formation in homogenates of seeds and seedlings of A. thaliana Col-0. Furthermore, we wanted to localize specifier proteins within the cell to enable a better understanding of their function. Another aim was to establish the interaction between myrosinase TGG1 and specifier proteins in planta. The main glucosinolate hydrolysis products in homogenates of A. thaliana Col-0 seeds and seedlings were the nitriles. The analysis of the T-DNA-mutants nsp2-1 and nsp2-2 showed that AtNSP2 is responsible for nitrile formation in seeds. In homogenates of seedlings of the nsp1-1 mutant, nitrile formation was reduced relative to the wildtype. This indicates a role of AtNSP1 for nitrile formation in seedlings. In addition, AtNSP3, AtNSP4 and AtNSP5 may contribute. To analyse their subcellular localisation, AtNSP1, AtNSP2, AtNSP4, AtNSP5, At3g07720 and the thiocyanate-forming protein of Thlaspi arvense were transiently expressed in Nicotiana benthamiana in fusion with the fluorescence protein Venus. All analysed proteins were localized in the cytoplasm. To analyse the interaction between TGG1 and AtNSP3 in planta by bimolecular fluorescence complementation, the proteins were produced in separate compartments of N. benthamiana-cells in fusion with the reporter-fragments VYNE and SCYCE and brought together through extraction. This approach failed because of a strong background-fluorescence. With mCherry as reporter, background-fluorescence was reduced, but signal indicating an interaction wasn’t detected. Cytosolic expression of myrosinase without signal peptide (TGG-oSP) allowed the analysis of unglycosylated myosinase. The experiments demonstrated an interaction between TGG1-oSP and AtNSP3. TGG1-oSP was also examined with AtNSP5 or At3g07720, but an interaction could not be demonstrated

NSP-Dependent Simple Nitrile Formation Dominates upon Breakdown of Major Aliphatic Glucosinolates in Roots, Seeds, and Seedlings of Arabidopsis thaliana Columbia-0

One of the best-studied plant defense systems, the glucosinolate-myrosinase system of the Brassicales, is composed of thioglucosides known as glucosinolates and their hydrolytic enzymes, the myrosinases. Tissue disruption brings these components together, and bioactive products are formed as a consequence of myrosinase-catalyzed glucosinolate hydrolysis. Among these products, isothiocyanates have attracted most interest as chemical plant defenses against herbivores and pathogens and health-promoting compounds in the human diet. Previous research has identified specifier proteins whose presence results in the formation of alternative product types, e.g., nitriles, at the expense of isothiocyanates. The biological roles of specifier proteins and alternative breakdown products are poorly understood. Here, we assessed glucosinolate breakdown product profiles obtained upon maceration of roots, seedlings and seeds of Arabidopsis thaliana Columbia-0. We identified simple nitriles as the predominant breakdown products of the major endogenous aliphatic glucosinolates in root, seed, and seedling homogenates. In agreement with this finding, genes encoding nitrile-specifier proteins (NSPs) are expressed in roots, seeds, and seedlings. Analysis of glucosinolate breakdown in mutants with T-DNA insertions in any of the five NSP genes demonstrated, that simple nitrile formation upon tissue disruption depended almost entirely on NSP2 in seeds and mainly on NSP1 in seedlings. In roots, about 70–80% of the nitrile-forming activity was due to NSP1 and NSP3. Thus, glucosinolate breakdown product profiles are organ-specifically regulated in A. thaliana Col-0, and high proportions of simple nitriles are formed in some parts of the plant. This should be considered in future studies on biological roles of the glucosinolate-myrosinase system

Public Painting - Malerei und Handlung

Das Magazin "Public Painting – Malerei und Handlung" versammelt künstlerischen Projekte, die auf unterschiedliche Weise Verhältnisse von Malerei zum öffentlichen Raum reflektieren, und Verhältnisse thematisieren, die im öffentlichen Raum ausagiert werden. Die Beiträge entstanden als Resultat der Kooperation des kunstwissenschaftlichen Seminars "Malerei als Handlung" von Anne Röhl und des kunstpraktischen Seminars "public painting" von Sebastian Freytag während des Sommersemesters 2021 an der Universität Siegen. Unter dem Titel "public painting" wurden Schnittstellen von malerischer Praxis und öffentlichen Raum diskutiert. Die Studierenden des Seminars "Malerei als Handlung" haben sich wiederum damit beschäftigt, wie die Gattung Malerei im Verlauf des 20. Jahrhunderts durch die Abkehr von traditionellen Entstehungsprozessen und Malhandlungen befragt wurde und skulptural und/oder performativ wird. Daneben widmeten sich die Studierenden des kunsthistorischen Seminars der schriftlichen Auseinandersetzung mit den künstlerischen Resultaten des kunstpraktischen Seminars. Die Auseinandersetzung mit dem öffentlichen Raum stellte im Sommersemester 2021 eine besondere Herausforderung dar, da er pandemiebedingt nur eingeschränkt nutzbar war. Die künstlerischen Ergebnisse dokumentieren somit auch diese Zeit

Scouting the Autonomous Agricultural Machinery Market

The development and introduction of machines ranging from highly automated to driverless will have a strong impact on global agriculture - there is no doubt about this. Today, fully autonomous tractors or combine harvesters are no longer prototypes, but are already in use in preliminary stages. This inevitably raises the question of how fast and to what extent the new technologies will determine the market in the future. To examine this question, Fraunhofer IESE in cooperation with the Kleffmann Group conducted this study, which deals with the state of the art as well as the future development of the autonomous agricultural machinery market. Several parallel paths were taken in the study: (1) The first task was to identify and classify the influencing factors related to the market. This is linked to the determination of changes in agricultural work processes within the framework of autonomous agricultural machinery development. (2) These influencing factors in combination with corresponding expert assessments as well as market data on tractor sales were then used within the framework of an empirical modeling approach to develop possible future scenarios up to the year 2045

RNA hydrolysis by heterocyclic amidines and guanidines: parameters affecting reactivity

2-Aminobenzimidazole 10, although a weak catalyst in the monomeric state, is a successful building block for effective artificial ribonucleases. In an effort to identify new building blocks with improved catalytic potential, RNA cleavage by a variety of heterocyclic amidines and guanidines has been studied. In addition to pKa values and steric effects, the energy difference between tautomeric forms seems to be another important parameter for catalysis. This information is available from quantum chemical calculations on higher levels, but semiempirical methods are sufficient to get a first estimate. According to this assumption, imidazoimidazol 18, characterized by isoenergetic tautomeric forms, is superior to 2-aminoimidazol 6, the best candidate among the simple compounds. By far the largest effects are seen with 2-aminoperimidine 24, which rapidly cleaves RNA even in the micromolar concentration range. The impressive reactivity, however, is related to a tendency of compound 24 to form polycationic aggregates which are the actual catalysts