No description available.

No description available.

Cyclooxygenase (COX) and lipoxygenase (LOX) catalyze the rate-determining step in the production of arachidonic acid- derived signaling molecules (eicosanoids) within the body. COX has been extensively investigated, which has enabled the design of non-steroidal inflammatory drugs (NSAIDs) such as aspirin, acetaminophen (ApAP) and ibuprofen. However, there are still fundamental questions surrounding the LOX family of enzymes, which has limited the development of isoform specific inhibitors. The structural basis and regio- and stereoselectivity of the LOX isoforms are not known. Herein, we describe two strategies to develop isoform-specific inhibitors of lipoxygenase. Efforts were focused on the synthesis of unnatural lipid derivatives, in which the methylene hydrogen atoms on the substrate were replaced with a moiety lacking a labile hydrogen atom, such as fluorine. This would allow the LOX enzyme to remain in an active form, while preventing enzyme turnover. This preliminary work will enable the assessment of their activity as inhibitors and attempts at their co-crystallization might provide the first insight into the binding mode of these fatty acid substrates. The preparation of a small library of acylated 2-aminopyrimidines and their efficacy as inhibitors of soybean lipoxygenase-1 was explored. Preliminary studies suggest the mode of action occurs through a bi-dentate coordination of the ferric iron atom. Modifications of the acylated 2-aminopyrimidines to make it more substrate-like and to increase its lipophilicity, yielded inhibitors with low micromolar IC50 values. With further optimization, acyl 2-aminopyrimidines could serve as a useful platform for the discovery of safe and efficient isoform specific inhibitors.

No description available.

An efficient approach to combat the accumulation of aflatoxin is the development of germplasm resistant to infection and spread of A. flavus in maize, one of the most important cereal grains in the world. Lipoxygenases (LOXs) are a group enzymes that catalyze oxygenation of polyunsaturated fatty acids (PUFAs). LOX derived oxilipins play critical roles in plant defense against pathogens such as A. flavus. The objectives of this study were to report sequence diversity and expression patterns for all LOX genes, and map their effect on aflatoxin accumulation via linkage and association mapping. Genes GRMZM2G102760 (ZmLOX 5) and GRMZM2G104843 (ZmLOX 8) fell under previously published QTL in one of four mapping populations and appear to have a measurable effect on the reduction of aflatoxin in maize grains. The association mapping result shows 19 of the total 215 SNPs found within the sequence of the ZmLOXs were associated with reduced aflatoxin levels.

The activity of plant 9-lipoxygenases (LOXs) influences the outcome of Arabidopsis thaliana interaction with pathogen and insects. Evidence provided here indicates that in Arabidopsis, 9-LOXs facilitate infestation by Myzus persicae, commonly known as the green peach aphid (GPA), a sap-sucking insect, and infection by the fungal pathogen Fusarium graminearum. in comparison to the wild-type plant, lox5 mutants, which are deficient in a 9-lipoxygenase, GPA population was smaller and the insect spent less time feeding from sieve elements and xylem, thus resulting in reduced water content and fecundity of GPA. LOX5 expression is induced rapidly in roots of GPA-infested plants. This increase in LOX5 expression is paralleled by an increase in LOX5-synthesized oxylipins in the root and petiole exudates of GPA-infested plants. Micrografting experiments demonstrated that GPA population size was smaller on plants in which the roots were of the lox5 mutant genotype. Exogenous treatment of lox5 mutant roots with 9-hydroxyoctadecanoic acid restored water content and population size of GPA on lox5 mutants. Together, these results suggest that LOX5 genotype in roots is critical for facilitating insect infestation of Arabidopsis. in Arabidopsis, 9-LOX function is also required for facilitating infection by F. graminearum, which is a leading cause of Fusarium head blight (FHB) disease in wheat and other small grain crops. Loss of LOX1 and LOX5 function resulted in enhanced resistance to F. graminearum infection. Similarly in wheat, RNA interference mediated silencing of the 9-LOX homolog TaLpx1, resulted in enhanced resistance to F. graminearum. Experiments in Arabidopsis indicate that 9-LOXs promote susceptibility to this fungus by suppressing the activation of salicylic acid-mediated defense responses that are important for basal resistance to this fungus. the lox1 and lox5 mutants were also compromised for systemic acquired resistance (SAR), an inducible defense mechanism that is systemically activated throughout a plant in response to a localized infection. the lox1 and lox5 mutants exhibited reduced cell death and delayed hypersensitive response when challenged with an avirulent strain of the bacterial pathogen Pseudomonas syringae pv tomato. LOX1 and LOX5 functions were further required for the synthesis as well as perception of a SAR-inducing activity present in petiole exudates collected from wild-type avirulent pathogen-challenged leaves. Taken together, results presented here demonstrate that 9-LOX contribute to host susceptibility as well as defense against different biotic stressors.

Die Lipidoxidation kann sowohl enzymatisch als auch nicht enzymatisch erfolgen. Der erste Schritt der enzymatischen Oxidation wird durch Lipoxygenasen katalysiert, von welchen es in Arabidopsis thaliana sechs verschiedene Isoformen gibt. Dabei werden die Lipoxygenasen nach dem Kohlenstoffatom klassifiziert, welches sie oxidieren. Somit gehören die LOX1 und LOX5 zu den 9-Lipoxygenasen, während LOX2, LOX3, LOX4 und LOX6 zu den 13 Lipoxygenasen zählen. Während der Samenalterung findet vermehrt eine Lipidperoxidation statt, welche mit einem Verfall des Samens sowie einer verringerten Keimrate korreliert. Im Rahmen dieser Arbeit wurde zunächst erfolgreich ein System zur künstlichen Samenalterung von Arabidopsis thaliana etabliert. Bei der künstlichen Alterung stiegen ähnlich wie bei der natürlichen Samenalterung oxidierte Lipide an und die Keimrate fiel ab. Nach Alterung konnte ein Anstieg von sechs verschiedenen oxidierten Triacylglycerolen detektiert werden. Es konnte in dieser Arbeit mit Hilfe von Mutanten mit Defekten in mehreren der Lipoxygenase Gene gezeigt werden, dass die Oxidation dieser veresterten Fettsäuren zum größten Teil nicht enzymatisch erfolgt. Bei der Alterung stiegen zudem enzymatisch gebildete 9 Lipoxygenase Produkte wie freie Hydroxy- und Ketofettsäuren an. Bei einer Analyse der freien oxidierten Fettsäuren konnte ebenfalls mit Lipoxygenase Mutanten ermittelt werden, dass diese hauptsächlich via LOX1 oxidiert werden. Die Untersuchung der Keimraten der Lipoxygenase Mutanten nach Alterung zeigte in mehreren Versuchen eine leicht erhöhte Keimrate der lox1 im Vergleich zum Wildtyp. Eine exogene Behandlung von Wildtyp Samen mit verschiedenen 9-Lipoxygenase Produkten, welche bei der Alterung ansteigen, führte allerdings nicht zu einer Keimungshemmung. Somit scheinen Produkte wie Hydroxy- und Ketofettsäuren der 9-Lipoxygenase LOX1 nicht die Hauptursache für die Keimungshemmung nach Alterung zu sein. Darüber hinaus konnte in dieser Arbeit gezeigt werden, dass eine Behandlung der Blüten des Wildtyps mit Methyljasmonat zu einer signifikant höheren Keimrate der Samen im Vergleich zu Samen von unbehandelten Pflanzen nach Alterung führt. Ein „Lipidprofiling“ der Samen von mit Methyljasmonat behandelten Pflanzen wies signifikant geringere Gehalte sowohl an freien als auch veresterten oxidierten Fettsäuren auf, was mit einer erhöhten Lebensfähigkeit korrelierte. Diese Erkenntnisse könnten von großer Relevanz für die Landwirtschaft sein, falls eine Übertragung auf Nutzpflanzen möglich ist. Ein weiterer Schwerpunkt dieser Arbeit war eine eingehende Untersuchung der Rolle und Funktion der LOX6. Mit Hilfe von GUS Färbungen konnte eine Lokalisation der LOX6 in Blättern und Wurzeln nachgewiesen werden. Zudem wurde ein 35SLOX6GFP Konstrukt erstellt und in Arabidopsis thaliana Pflanzen stabil transformiert. Mit den selektionierten Linien könnte in Zukunft auch die intrazelluläre Lokalisation der LOX6 untersucht werden. Außerdem wurden Konstrukte mit dem Reportergen GFP und AOS sowie LOX2 hinter dem 35S Promotor kloniert, welche ebenfalls für weitere Lokalisations- und Kolokalisationsstudien genutzt werden können. Zudem wurde mit der Klonierung eines Konstruktes begonnen, um in Zukunft einen spezifischen LOX6 Antikörper herstellen und auch die endogene LOX6 Lokalisation in dem Wildtyp analysieren zu können. Um die Produkte der LOX6 zu untersuchen, wurden 35SLOX6 Linien sowie die lox6 Mutante verwendet. Obwohl Hydroxyfettsäuren und Jasmonate Folgeprodukte der LOX6 sind, wiesen die 35SLOX6 Linien weder basal, noch nach Stress erhöhte Gehalte dieser im Vergleich zum Wildtyp auf. Somit geben die 35SLOX6 Linien einen Hinweis darauf, dass LOX6 im Wildtyp nicht limitierend für die Produktion von Hydroxyfettsäuren und Jasmonaten sein könnte. Um zu untersuchen, ob das Substrat der LOX6 der limitierende Faktor sein könnte, wurde eine Behandlung mit α Linolensäure durchgeführt. Dabei entstanden allerdings nicht mehr Folgeprodukte der LOX6, sondern es fand sowohl in den 35SLOX6 Linien als auch in dem Wildtyp eine massive nicht enzymatische radikalische Oxidation der Fettsäuren statt. Um festzustellen, ob sich durch eine LOX6 Überexpression das Metabolom ändert, wurde eine „untargeted Analyse“ mit 35SLOX6 Linien durchgeführt. Diese zeigte vier Metabolite, welche in den 35SLOX6 Linien im Vergleich zum Wildtyp unterschiedlich stark vorhanden waren. Zudem sollte untersucht werden, ob sich die Physiologie und Stressresistenz in den Überexpressionslinien im Vergleich zum Wildtyp unterscheiden. Dabei zeichneten sich die 35SLOX6 Linien durch kleinere, hellere und rundere Blätter aus. Zudem wurden die Wurzeln der 35SLOX6 Linien bei Fraßversuchen mit Pocellio scaber im Vergleich zum Wildtyp weniger bevorzugt gefressen. Diese Erkenntnisse sowie die generierten Konstrukte und Pflanzenlinien können in der Zukunft einen weiteren Einblick in die vielfältigen Funktionen und Produkte der LOX6 gewähren. / Lipidoxidation can take place enzymatically and non-enzymatically. The first step of the enzymatic oxidation is catalysed via lipoxygenases. In Arabidopsis thaliana there are six lipoxygenase isoforms. The lipoxygenases are characterized by the carbon atom they oxidise. LOX1 and LOX5 are 9-lipoxygenases, while LOX2, LOX3, LOX4 and LOX6 are 13 lipoxygenases. During seed ageing lipid peroxidation takes place, which correlates with a deterioration of the seed and a lower germination rate. First, a method for artificial ageing of Arabidopsis thaliana seeds was successfully established as a part of this work. During artificial seed ageing, oxidised lipids increased and the germination rate decreased similar to natural ageing. During artificial ageing an accumulation of six oxidised triacylglycerols could be detected. In this work, it could be shown with the help of mutants with defects in the lipoxygenase genes, that the oxidation of esterified fatty acids mainly takes place non-enzymatically. Moreover, enzymatically formed free 9-lipoxygenase products such as hydroxy and keto fatty acids increase during the process of ageing. An analysis of the free fatty acids in lipoxygenase mutants lead to the conclusion that they are formed primarily by LOX1. The lox1 mutant showed a slightly higher germination rate than the wild type after seed ageing in the majority of the experiments. However, an exogenous treatment of wild type seeds with free 9-lipoxygenase products, which increase during ageing, did not inhibit the germination rate. Therefore, LOX1 (9-lipoxygenase) products like hydroxy and keto fatty acids do not seem to be the main cause for the inhibition of germination after ageing. In addition, this work shows that a methyl jasmonate treatment of wild type flowers leads to a significant higher germination rate of their seeds after ageing in comparison to the seeds of untreated wild type plants. A lipid profiling revealed significantly lower levels of oxidised esterified as well as free fatty acids after ageing in seeds of treated wild type plants compared to untreated ones, which correlates with a higher germination rate. These findings could be of great value for the agriculture if they are transferable to crop plants. Another focus of this work was set on investigating the function and relevance of LOX6. The localization of LOX6 in leaves and roots could be confirmed with the help of GUS stainings. Furthermore, a 35SLOX6GFP construct was generated and stably transformed in Arabidopsis thaliana plants. With the selected lines it will be possible to investigate the intracellular localization of LOX6 in the future. Moreover, constructs with the reporter gene GFP and AOS or LOX2 were cloned behind the 35S promoter which can also be used for additional localization and co-localization experiments. To analyse the endogenous localization of LOX6 in the wild type, the cloning of a construct was started to generate a specific antibody in the future. To investigate the different products of LOX6, 35SLOX6 lines and the lox6 mutant were used. Although hydroxy fatty acids and jasmonates are secondary products of LOX6, neither basal nor after different stress treatments elevated levels could be detected in the overexpression lines compared to the wild type. This finding indicates that LOX6 may not be limiting for the production of jasmonates and hydroxy fatty acids in the wild type. Moreover, to investigate if the substrate of LOX6 could be a limiting factor, a treatment with α linolenic acid was performed. However, this did not lead to more LOX6 secondary products but rather to a massive increase of non-enzymatic radical triggered oxidation of fatty acids in the 35SLOX6 lines as well as in the wild type. To examine whether an overexpression of LOX6 leads to changes in the metabolom, an untargeted analysis with 35SLOX6 lines was performed. This analysis revealed four metabolites, which were present in different amounts in 35SLOX6 lines and the wild type. Apart from that, the physiology and stress resistance of the 35SLOX6 lines should be investigated for differences compared to the wild type. The overexpression lines exhibited smaller, rounder and paler leaves. In feeding experiments, the roots of 35SLOX6 plants were less attractive to the rough woodlouse Porcellio scaber than the wild type. The insights of this work, together with the generated constructs and plant lines could help to gain a better understanding of the versatile functions and products of LOX6 in the future.

Plant lipoxygenases (LOX) are non-heme iron containing dioxygenases that catalyze the hydroperoxidation of polyunsaturated fatty acids resulting in the synthesis of a large number of functionally diverse oxylipins. Although the physiological functions of jasmonate-producing 13-LOXs in dicots have been reported and highlighted in host defenses to pathogens and insects, the functions of 9-LOXs and 9-LOX derived oxylipins remain obscure for both monocots and dicots. The objective of this study was to elucidate the biochemical, molecular and physiological roles of a segmentaly duplicated pair of 9-LOXs, ZmLOX4 and ZmLOX5, in host defenses to diverse stresses. Despite of their extreme similarities at the sequence levels, the ZmLOX4 was preferentially expressed in underground organs, whereas ZmLOX5 was stress-induced in aboveground organs. Both genes were highly induced by exogenous jasmonic acid (JA) but transcripts of ZmLOX5 only were strongly induced in wounded leaves as well as in response to insect infestation, suggesting the role of ZmLOX5 in plant resistance response against insect herbivory. To test potential function of ZmLOX4 and ZmLOX5, near-isogenic wild-type and mutants were generated. In this study, I provided genetic evidence that ZmLOX5 is involved in host defense against insect herbivores via the regulation of wound-induced JA biosynthesis. Contrary to the role in insect defenses, ZmLOX5 mediated metabolism contributes to enhanced susceptibility to a leaf fungal pathogen, Colletotricum graminicola. ZmLOX4 appears to have evolved a defense function against C. graminicola. In addition, lox4 and lox5 mutants have opposite phenotypes in their ability to support production of conidia and to facilitate colonization of kernels in response to Aspergillus flavus. However, the two mutants were similar to each other in their enhanced susceptibility to kernel colonization and conidia production of Fusarium verticillioides. In conclusion, the data suggest that these two 9-LOXs, ZmLOX4 and ZmLOX5 and their metabolites have distinct roles in plant-insect and plant-pathogen interactions.

Universiẗat, Diss., 2007--Frankfurt (Main). / Zsfassung in dt. und engl. Sprache.

Univ., Diss., 2009--Frankfurt (Main).