O advento de técnicas de sequenciamento de alta eficiência possibilitou o estudo mais aprofundado de pequenos RNAs, como os microRNAs (miRNAs), a classe melhor caracterizada, e a identificação de novas classes como a dos transfer RNA-derived Fragments (tRFs). Os pequenos RNAs podem atuar como reguladores negativos da expressão gênica do seu transcrito alvo. Este mecanismo, denominado Silenciamento Gênico Pós-Transcricional (PTGS) ou RNA interferência (RNAi), pode ocorrer pela indução da clivagem do transcrito alvo, ou pela repressão da tradução do mesmo. Em soja, ainda não foram descritos miRNAs atuantes na germinação da semente, os quais foram abordados no primeiro capítulo desta tese. Utilizando duas bibliotecas de sequenciamento de alta eficiência, uma relativa a sementes maduras e outra composta de uma combinação de sementes em germinação (3, 5 e 7 dias), foram identificados um total de 178 microRNAs, sendo 36 inéditos. Dos 178, 8 miRNAs com alvos potencialmente relacionados à germinação da semente, às rotas de auxina, giberelina, metabolismo lipídico, de nitrogênio e homeostase de potencial redox, foram validados por análise de degradoma. O segundo capítulo aborda a caracterização de tRFs em Arabidopsis associados com proteínas Argonauta (AGO), as quais são essenciais ao RNAi. Foram utilizadas 26 bibliotecas de sequenciamento de argonautas imunoprecipitadas (AGO-IP), relativas às AGOs 1, 2, 4, 5, 7 e 9, além de 3 bibliotecas de degradoma. O mapeamento destas sequências nos tRNAs de Arabidopsis revelou que estes pequenos RNAs são majoritariamente associados a AGO1 e 2, sendo a classe 5' de 19 nucleotídeos de comprimento a mais comum. Contudo, estes não obedecem aos critérios de direcionamento a proteínas AGO relativos ao primeiro nucleotídeo do pequeno RNA, como ocorre com miRNAs. Foram identificados quatro transcritos alvos, validados por análise do degradoma, os quais possivelmente sofrem PTGS via tRFs. Ambos os capítulos apresentam uma robusta caracterização in silico de pequenos RNAs em plantas inferindo suas possíveis funções. Contudo, mais experimentos devem ser efetuados para confirmação de seus papeis em soja e Arabidopsis. / The advent of the deep sequencing approach enabled a better characterization of small RNAs, such as microRNAs (miRNAs), the well-known small RNA class, and the identification of new classes like the transfer RNA-derived Fragments (tRFs). The small RNAs can act as negative regulators of gene expression of their target transcript. This mechanism, known as Post Transcriptional Gene Silencing (PTGS), involves dicing of the target transcript, or translational repression. In soybean, the microRNAs acting on seed germination are unknown. These miRNAs were described in the first chapter of this thesis. Using two deep sequencing libraries, relative to the mature seeds and a combination of germinating seeds (3, 5 and 7 days). A total of 178 miRNAs were identified, including 36 new ones. Eight miRNAs had targets potentially related to seed germination including some acting on auxin and gibberellin pathways, lipid and nitrogen metabolism and redox homeostasis, and were validated by degradome analysis. The second chapter showed the characterization of Argonaut (AGO) associated tRFs in Arabidopsis. AGO is an essential protein for PTGS. A total of 26 deep sequencing libraries from immunoprecipitated Argonauts (AGO-IP), relative to the AGO 1, 2, 4, 7, and 9, plus 3 degradome libraries were used. The tRFs were mainly associated with AGO1 and 2, and the 5' class of 19 nucleotides in length was the most common one. However the tRFs did not follow the rule for AGO loading, were the first nucleotide lead the microRNA to a specific AGO. We identified four tRF target transcripts validated by degradome analysis, which possibly undergo the PTGS pathway. Both chapters present a robust in silico characterization of small RNAs in plants, inferring their possible functions. However, more experiments should be performed to confirm their roles in soybean and Arabidopsis.

M.Sc. (Biochemistry) / Plants are constantly exposed to a range of environmental stresses which can be biotic or abiotic in nature. These stresses/threats result in cross-talk between signaling pathways which trigger numerous defense responses. These reactions include activation of defense genes, accumulation of reactive oxygen species (ROS) and biosynthesis of small protective/defensive chemical compounds. The plant metabolome is comprised of primary and secondary metabolites, and while primary metabolites are involved in crucial metabolic processes such as growth and development, the latter play a key role in plant-pathogen interactions (defense). Metabolomics is one of the most recent “omic” technologies and involves the study of metabolites and their metabolic pathways under certain physiological conditions. This provides biological knowledge about the system under study giving insight into the cellular processes that define the phenotype of a cell, tissue or whole organism. In the present study a metabolomic approach was used to elucidate and analyze changes in the metabolism of Arabidopsis thaliana cells and leaves following lipopolysaccharide (LPS) treatment. Camalexin (a phytoalexin) and a group of metabolites known as glucosinolates (phytoanticipins) have been shown to accumulate in response to plant-pathogen and plant-herbivore interactions and were the main focus of the study. A number of studies involving herbivore-induced glucosinolate production have been conducted; however, in terms of microbial attack, studies are limited. The following study therefore provided insight into the effect that LPS treatment has on the biosynthetic pathways for indolic, aliphatic and aromatic glucosinolates....

Reaktive elektrophile Spezies-Oxylipine (RES-Oxylipine) finden sich in Pflanzen- und Tierzellen und zeichnen sich durch eine für sie typische Anordnung von Atomen aus: einer α,β ungesättigten Carbonyl Gruppe. In Pflanzenzellen gehören unter anderem 2-(E)-Hexenal und die Vorstufe der Jasmonsäure 12-Oxophytodiensäure (OPDA) zu den RES-Oxylipinen, in Tierzellen z.B. Prostaglandin A1 (PGA). RES-Oxylipine üben Signalfunktionen aus, wie dies in Pflanzenzellen funktioniert ist jedoch noch nicht bekannt. Ziel dieser Arbeit ist dabei einen möglichen RES-Oxylipin Signalweg aufzuklären und die beteiligten Gene zu identifizieren. Es konnte aber gezeigt werden, dass die Expressionsrate von bestimmten Genen wie z.B. GST6 durch RES-Oxylipine spezifisch induziert wird. Zur Untersuchung des RES-Oxylipin Signalweges wurde der GST6 Promotor vor das Luciferase-Gen fusioniert, um so ein RES-Oxylipin spezifisches Reportersystem zu erhalten. Die Ethylmethansulfonat mutagenisierten Linien wurden auf geänderte Luciferase-Aktivität hin untersucht. Dabei wurden drei Mutanten isoliert, die in dieser Arbeit näher untersucht wurden. Eine zeigte basal erhöhte Luciferase-Aktivität (constitutive overexpresser 3 = coe3) und die anderen beiden erniedrigte Luciferase-Aktivität nach PGA Gabe (non responsive 1 und 2 = nr1 und nr2). In dieser Arbeit konnte gezeigt werden, dass die Phänotypen in allen 3 Mutanten rezessiv vererbt werden und die Mutanten nicht zueinander allel sind. Zudem war die veränderte Luciferase-Aktivität nicht durch geänderte Phytohormonspiegel oder durch Mutationen im GST6 Promotor erklärbar. Auf die Gabe von RES, wie Benzylisothiocyanat oder Sulforaphan, sowie auf endogene RES-Oxylipine, wie OPDA und Hexenal, reagierten die Mutanten auf ähnliche Weise, wie nach PGA Gabe. Weiterführende Untersuchungen zeigten, dass sich die drei Mutanten stark voneinander unterschieden. Das Transkriptom kontrollbehandelter coe3 Pflanzen unterschied sich stark von dem der GST6::LUC Pflanzen. Die Mutante war trockenstressresistenter zudem war sie sensibler gegenüber NaCl, was jedoch nicht von einer veränderten Reaktion auf Abscisinsäure herrührte. Des Weiteren war der Chlorophyllabbau bei dunkel inkubierten Blättern geringer. Bei der Lokalisierung der Mutation, die noch nicht abgeschlossen ist, konnten Chromosom 2 und 5 als die wahrscheinlichsten Kandidaten ermittelt werden. Weitere Analysen sind nötig um den Bereich weiter eingrenzen zu können. Die Mutante nr1, die sich durch verminderte Reaktion auf RES-Oxylipine auszeichnete, zeigte einen kleineren Wuchs und ein deutlich verzögertes Blühen. Außerdem wies die Mutante erhöhte Argininspiegel in ihren Blättern auf. Das Transkriptom unterschied sich sowohl bei kontrollbehandelten, als auch bei PGA behandelten nr1 Pflanzen massiv von denen der gleichbehandelten Kontrollen. Auch die nr1 schien trockenstressresistenter zu sein, sie war im Gegensatz zur coe3 aber robuster gegenüber höheren Konzentrationen an NaCl. Mit Hilfe eines „Next Generation Genome-Mappings“ war es möglich die Mutation am Ende von Chromosom 3 zu lokalisieren und auf fünf mögliche Gene einzugrenzen. Weitere Untersuchungen müssen nun klären, welches dieser Gene ursächlich für den Phänotyp der geänderten Luciferase-Aktivität ist. Die zweite Mutante mit einer reduzierten Reaktion auf RES-Oxylipine war die nr2. Überraschender Weise unterschied sich das Transkriptom kontrollbehandelter nr2 Pflanzen deutlich stärker von dem der gleichbehandelten GST6::LUC Pflanzen, als das nach PGA Gabe der Fall war. Sie reagierte nur mit sehr schwacher Luciferase-Aktivität auf Verwundung und war zudem deutlich sensibler gegenüber Trockenheit. Für eine zukünftige Lokalisation der ursächlichen Mutation wurden entsprechende Kreuzungen durchgeführt aus deren Samen jederzeit mit einer Selektionierung begonnen werden kann. Mit dieser Arbeit konnte ein erster großer Schritt in Richtung Identifikation der, für die geänderte Luciferase-Aktivität, verantwortlichen Mutation gemacht werden, sowie erste Reaktionen der Mutanten auf abiotische Stressfaktoren untersucht werden. Somit ist man der Entdeckung von Signaltransduktionsfaktoren, die RES-Oxylipinabhängig reguliert werden, einen wichtigen Schritt näher gekommen. / Reactive electrophilic species oxylipins (RES-oxylipins) can be found in plant and animal cells and contain an α,β unsaturated carbonyl group. In plant cells 2-(E)-hexenal and 12-oxo-phytodienoic acid (OPDA), the precursor of jasmonic acid, belong to the RES-oxylipins, in animal cells prostaglandin A1 (PGA). RES-oxylipins play an important role in signal transduction but it is still unknown how this functions in plant cells. In previous publications, it could be shown, that RES-oxylipins induce the expression of certain genes like GST6 specifically. To further investigate the possibility of a RES-oxylipin pathway the GST6 promotor was fused to the luciferase gene to get a RES-oxylipin sensitive reporter system. The ethyl methanesulfonate (EMS) mutagenized lines were screened for altered luciferase activity under basal conditions and after treatment with PGA. Three lines were selected for further investigation: one with a constitutive higher luciferase activity (constitutive overexpresser 3 = coe3) and two with a reduced luciferase activity after PGA treatment (non responsive 1 and 2 = nr1 and nr2). In this thesis, it could be shown, that the mutation, which is responsible for the altered luciferase activity, is recessive and not allelic to each other. Furthermore, neither altered phytohormone levels nor mutations in the GST6 promotor are responsible for the changes in the luciferase activity. The response of these mutants to RES, like benzyl isothiocyanate (BITC) or sulforaphane, or endogenous RES-oxylipins, like OPDA or hexenal, is comparable to the response to PGA treatment. Further investigations show huge differences between the mutants. Control treated coe3 plants had very different transcriptomes compared to the control line. The coe3 mutant was more resistant to drought stress but more susceptible to salt compared to the control. This was not due to a changed response to abscisic acid (ABA). Another observed phenotype was the reduced chlorophyll depletion in dark incubated leaves. The localization of the mutation could not be completed within this thesis but chromosome 2 and 5 could be identified as most likely positions. Further investigations on this topic are needed to complete the localization. The nr1 mutant showed a reduced growth and delayed flowering phenotype and higher arginine levels could be detected in the leaves. The transcriptome exhibited huge differences after both control treatment and PGA treatment compared to the GST6::LUC. In drought experiments, the nr1 was also more resistant but, compared to the coe3, also more robust against higher salt concentrations. By next generation genome mapping it was possible to locate the mutation, which was responsible for the changes in luciferase activity after PGA treatment, at the end of chromosome 3. So there are five genes left who might be responsible for the observed phenotypes. Further investigations have to show which one is the one causing the phenotype. The nr2, as the third mutant investigated in this thesis showed highest differences to the GST6::LUC line after control treatment. It only had weak luciferase activity after wounding and was more susceptible to drought then the control. For further mapping experiments of the mutation in the nr2 mutant, F2 lines were generated. These are now ready to use to set up a mapping population. With this thesis it was possible to provide a milestone in identification of the mutations responsible for the changes in luciferase activity and in investigation of different abiotic stress responses. Now we are one step closer to discover signal transduction factors which are regulated by RES-oxylipins.

The involvement of several genes in DNA repair in animals and bacteria, and the induced expression of At3G02400 by Mitomycin C, suggest that this gene is involved in DNA repair in plants. I have tested this hypothesis by studying the regulation of its expression and identifying the function of this gene. To study the expression of this gene, I used a reporter line and bio-computing tools. To identify the function of the gene, I have isolated two T-DNA mutants and screened Targeting Induced Local Lesions in Genomes (TILLING) mutants. T-DNA mutagenesis is the process of inserting transfer DNA into the genome, which disrupts the genetic expression. My study showed that Camptothecin, Bleomycin, and Mitomycin C induced the expression of At3G02400. In addition to this, the promoter truncation studies identified a 50 bp region just upstream of the start codon of At3G02400 that maybe required for the induction of expression.

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published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

Les nématodes phytoparasites constituent une menace réelle pour la production alimentaire mondiale. Ils sont responsables de 14% des pertes de rendement de la production alimentaire globale, ce pourcentage est l’équivalent de 100 milliards de dollar américain par an. La lutte contre ces phytoparasites est devenue un défit majeur, notamment après l'interdiction de l'utilisation du nématicide le plus efficace, en raison de ses effets nocifs sur l’environnement. Par conséquent, des nouvelles sources pour la gestion des nématodes phytoparasites sont nécessaires et urgentes. Ce travail vise à déterminer si la plante modèle Arabidopsis thaliana pourrait servir comme une source naturelle de gènes de la résistance aux nématodes phytoparasites. Parmi des accessions d’Arabidopsis, on a trouvé une variation génétique naturelle de la résistance contre les nématodes à galles Meloidogyne javanica, on a également identifié plusieurs QTL de résistance aux nématodes, et finalement, on a réalisé une cartographie fine d’un des QTL détectés. / Plant-parasitic nematodes are a serious threat for global food production. They are responsible for 14% of global yield loss, equivalent to an economic value of more than 100 billion US$ per year. Pest management is challenging, in particular since the most efficient nematicide has been banned due to its devastating effect on the environment. Hence, novel sources for nematode management are urgently required. This work investigates whether the model plant Arabidopsis thaliana could serve as a natural source for resistance genes against plant-parasitic nematodes. It finds natural genetic variation among Arabidopsis accessions for resistance against the root-knot nematode Meloidogyne javanica, identifies several QTL for nematode resistance, and fine-maps one of these resistance QTL.

Thesis (Ph. D.)--Swedish University of Agricultural Sciences, 2001. / Includes bibliographical references.