Investigation of the metabolism of rare nucleotides in plants

Publikation: Qualifikations-/StudienabschlussarbeitDissertation

Autoren

  • Henryk Straube

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Details

OriginalspracheEnglisch
QualifikationDoctor rerum naturalium
Gradverleihende Hochschule
Betreut von
Datum der Verleihung des Grades9 Sept. 2022
ErscheinungsortHannover
PublikationsstatusVeröffentlicht - 2023

Abstract

Nukleotide sind Metaboliten, die am Primärstoffwechsel und an spezialisierten Stoffwechselvorgängen beteiligt sind und eine regulierende Rolle bei verschiedenen biochemischen Reaktionen in allen Lebensformen spielen. Während bei anderen Organismen das Nukleotidmetabolom umfassend charakterisiert wurde, ist in Pflanzen vergleichsweise wenig über die zellulären Konzentrationen von Nukleotiden bekannt. Ziel dieser Dissertation war es, das Nukleotidmetabolom und die Enzyme zu untersuchen, die die Zusammensetzung und Menge der Nukleotide in Pflanzen beeinflussen. Zu diesem Zweck wurde eine Methode zur Analyse von Nukleotiden und Nukleosiden in Pflanzen und Algen entwickelt (Kapitel 2.1), die ein effizientes Stoppen enzymatischer Aktivität, eine Flüssig-Flüssig-Extraktion und eine Festphasenextraktion unter Verwendung eines schwachen Ionenaustauschers umfasst. Mit dieser Methode konnte das Nukleotidmetabolom von Pflanzen eingehend analysiert werden, einschließlich der Quantifizierung von Desoxyribonukleotiden und Desoxyribonukleosiden mit geringer Abundanz. Die Einzelheiten der Methode wurden in einem Artikel zusammengefasst, der als Laborprotokoll dient (Kapitel 2.2). Darüber hinaus wurde ein Übersichtsartikel (Kapitel 2.3) verfasst, der die Literatur über die Analyse von Nukleotiden und die jüngsten technologischen Fortschritte zusammenfasst. Der Schwerpunkt lag hierbei auf Pflanzen und Faktoren, die die Analyse von Nukleotiden in Pflanzen beeinflussen oder behindern, d. h. eine komplexe Matrix, hochstabile Phosphatasen und physikalisch-chemische Eigenschaften von Nukleotiden. Um die subzellulären Konzentrationen von Metaboliten zu analysieren, wurde ein Protokoll für die schnelle Isolierung hochreiner Mitochondrien unter Verwendung einer Affinitätschromatographie entwickelt (Kapitel 2.4). Die Methode zur Analyse von Nukleotiden trug außerdem zu einer umfassenden Analyse des Nukleotidmetaboloms in keimenden Samen und in sich etablierenden Keimlingen von A. thaliana bei, wobei der Schwerpunkt auf Genen lag, die an der Synthese von Thymidilaten beteiligt sind (Kapitel 2.5), sowie zu der Charakterisierung eines neuen Enzyms des Purinnukleotidabbaus, der XANTHOSINE MONOPHOSPHATE PHOSPHATASE (Kapitel 2.6). Eine Proteinhomologieanalyse, die A. thaliana, S. cerevisiae und H. sapiens miteinander verglich führte zur Identifizierung und Charakterisierung eines Enzyms, das an der Reparatur von geschädigten Metaboliten in Pflanzen beteiligt ist, der INOSINE TRIPHOSPHATE PYROPHOSPHATASE (Kapitel 2.7). Es konnte gezeigt werden, dass dieses Enzym desaminierte Purinnukleotidtriphosphate dephosphoryliert und so deren Einbau in Nukleinsäuren verhindert. Funktionsverlustmutanten altern früh und weisen einen konstitutiv erhöhten Gehalt an Salicylsäure auf. Außerdem wurde die Quelle der desaminierten Purinnukleotide in Pflanzen untersucht, und es wurde gezeigt, dass abiotische Faktoren zur Nukleotidschädigung beitragen.

Zitieren

Investigation of the metabolism of rare nucleotides in plants. / Straube, Henryk.
Hannover, 2023. 241 S.

Publikation: Qualifikations-/StudienabschlussarbeitDissertation

Straube, H 2023, 'Investigation of the metabolism of rare nucleotides in plants', Doctor rerum naturalium, Gottfried Wilhelm Leibniz Universität Hannover, Hannover. https://doi.org/10.15488/13270
Straube, H. (2023). Investigation of the metabolism of rare nucleotides in plants. [Dissertation, Gottfried Wilhelm Leibniz Universität Hannover]. https://doi.org/10.15488/13270
Straube H. Investigation of the metabolism of rare nucleotides in plants. Hannover, 2023. 241 S. doi: 10.15488/13270
Straube, Henryk. / Investigation of the metabolism of rare nucleotides in plants. Hannover, 2023. 241 S.
Download
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title = "Investigation of the metabolism of rare nucleotides in plants",
abstract = "Nucleotides are metabolites involved in primary metabolism, and specialized metabolism and have a regulatory role in various biochemical reactions in all forms of life. While in other organisms, the nucleotide metabolome was characterized extensively, comparatively little is known about the cellular concentrations of nucleotides in plants. The aim of this dissertation was to investigate the nucleotide metabolome and enzymes influencing the composition and quantities of nucleotides in plants. For this purpose, a method for the analysis of nucleotides and nucleosides in plants and algae was developed (Chapter 2.1), which comprises efficient quenching of enzymatic activity, liquid-liquid extraction and solid phase extraction employing a weak-anionexchange resin. This method allowed the analysis of the nucleotide metabolome of plants in great depth including the quantification of low abundant deoxyribonucleotides and deoxyribonucleosides. The details of the method were summarized in an article, serving as a laboratory protocol (Chapter 2.2). Furthermore, we contributed a review article (Chapter 2.3) that summarizes the literature about nucleotide analysis and recent technological advances with a focus on plants and factors influencing and hindering the analysis of nucleotides in plants, i.e., a complex metabolic matrix, highly stable phosphatases and physicochemical properties of nucleotides. To analyze the sub-cellular concentrations of metabolites, a protocol for the rapid isolation of highly pure mitochondria utilizing affinity chromatography was developed (Chapter 2.4). The method for the purification of nucleotides furthermore contributed to the comprehensive analysis of the nucleotide metabolome in germinating seeds and in establishing seedlings of A. thaliana, with a focus on genes involved in the synthesis of thymidilates (Chapter 2.5) and the characterization of a novel enzyme of purine nucleotide degradation, the XANTHOSINE MONOPHOSPHATE PHOSPHATASE (Chapter 2.6). Protein homology analysis comparing A. thaliana, S. cerevisiae, and H. sapiens led to the identification and characterization of an enzyme involved in the metabolite damage repair system of plants, the INOSINE TRIPHOSPHATE PYROPHOSPHATASE (Chapter 2.7). It was shown that this enzyme dephosphorylates deaminated purine nucleotide triphosphates and thus prevents their incorporation into nucleic acids. Lossof-function mutants senesce early and have a constitutively increased content of salicylic acid. Also, the source of deaminated purine nucleotides in plants was investigated and it was shown that abiotic factors contribute to nucleotide damage.",
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N2 - Nucleotides are metabolites involved in primary metabolism, and specialized metabolism and have a regulatory role in various biochemical reactions in all forms of life. While in other organisms, the nucleotide metabolome was characterized extensively, comparatively little is known about the cellular concentrations of nucleotides in plants. The aim of this dissertation was to investigate the nucleotide metabolome and enzymes influencing the composition and quantities of nucleotides in plants. For this purpose, a method for the analysis of nucleotides and nucleosides in plants and algae was developed (Chapter 2.1), which comprises efficient quenching of enzymatic activity, liquid-liquid extraction and solid phase extraction employing a weak-anionexchange resin. This method allowed the analysis of the nucleotide metabolome of plants in great depth including the quantification of low abundant deoxyribonucleotides and deoxyribonucleosides. The details of the method were summarized in an article, serving as a laboratory protocol (Chapter 2.2). Furthermore, we contributed a review article (Chapter 2.3) that summarizes the literature about nucleotide analysis and recent technological advances with a focus on plants and factors influencing and hindering the analysis of nucleotides in plants, i.e., a complex metabolic matrix, highly stable phosphatases and physicochemical properties of nucleotides. To analyze the sub-cellular concentrations of metabolites, a protocol for the rapid isolation of highly pure mitochondria utilizing affinity chromatography was developed (Chapter 2.4). The method for the purification of nucleotides furthermore contributed to the comprehensive analysis of the nucleotide metabolome in germinating seeds and in establishing seedlings of A. thaliana, with a focus on genes involved in the synthesis of thymidilates (Chapter 2.5) and the characterization of a novel enzyme of purine nucleotide degradation, the XANTHOSINE MONOPHOSPHATE PHOSPHATASE (Chapter 2.6). Protein homology analysis comparing A. thaliana, S. cerevisiae, and H. sapiens led to the identification and characterization of an enzyme involved in the metabolite damage repair system of plants, the INOSINE TRIPHOSPHATE PYROPHOSPHATASE (Chapter 2.7). It was shown that this enzyme dephosphorylates deaminated purine nucleotide triphosphates and thus prevents their incorporation into nucleic acids. Lossof-function mutants senesce early and have a constitutively increased content of salicylic acid. Also, the source of deaminated purine nucleotides in plants was investigated and it was shown that abiotic factors contribute to nucleotide damage.

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