Investigation of the metabolism of rare nucleotides in plants

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.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

An inosine triphosphate pyrophosphatase safeguards plant nucleic acids from aberrant purine nucleotides

In plants, inosine is enzymatically introduced in some tRNAs, but not in other RNAs or DNA. Nonetheless, our data show that RNA and DNA from Arabidopsis thaliana contain (deoxy)inosine, probably derived from nonenzymatic adenosine deamination in nucleic acids and usage of (deoxy)inosine triphosphate (dITP and ITP) during nucleic acid synthesis. We combined biochemical approaches, LC–MS, as well as RNA-Seq to characterize a plant INOSINE TRIPHOSPHATE PYROPHOSPHATASE (ITPA) from A. thaliana, which is conserved in many organisms, and investigated the sources of deaminated purine nucleotides in plants. Inosine triphosphate pyrophosphatase dephosphorylates deaminated nucleoside di- and triphosphates to the respective monophosphates. ITPA loss-of-function causes inosine di- and triphosphate accumulation in vivo and an elevated inosine and deoxyinosine content in RNA and DNA, respectively, as well as salicylic acid (SA) accumulation, early senescence, and upregulation of transcripts associated with immunity and senescence. Cadmium-induced oxidative stress and biochemical inhibition of the INOSINE MONOPHOSPHATE DEHYDROGENASE leads to more IDP and ITP in the wild-type (WT), and this effect is enhanced in itpa mutants, suggesting that ITP originates from ATP deamination and IMP phosphorylation. Inosine triphosphate pyrophosphatase is part of a molecular protection system in plants, preventing the accumulation of (d)ITP and its usage for nucleic acid synthesis

Complexome profiling reveals association of PPR proteins with ribosomes in the mitochondria of plants

Mitochondrial transcripts are subject to a wealth of processing mechanisms including cis- and trans-splicing events, as well as base modifications (RNA editing). HUndreds of proteins are required for these processes in plant mitochondria, many of which belong to the pentatricopeptide repeat (PPR) protein superfamily. The structure, localization, and function of these proteins is only poorly Understood. Here we present evidence that several PPR proteins are boUnd to mitoribosomes in plants. A novel complexome profiling strategy in combination with chemical crosslinking has been employed to systematically define the protein constituents of the large and the small ribosomal subunits in the mitochondria of plants. We identified more than 80 ribosomal proteins, which include several PPR proteins and other non-conventional ribosomal proteins. These findings reveal a potential coupling of transcriptional and translational events in the mitochondria of plants. Furthermore, the data indicate an extremely high molecular mass of the “small” subunit, even exceeding that of the “large” subunit

Initiation of cytosolic plant purine nucleotide catabolism involves a monospecific xanthosine monophosphate phosphatase

In plants, guanosine monophosphate (GMP) is synthesized from adenosine monophosphate via inosine monophosphate and xanthosine monophosphate (XMP) in the cytosol. It has been shown recently that the catabolic route for adenylate-derived nucleotides bifurcates at XMP from this biosynthetic route. Dephosphorylation of XMP and GMP by as yet unknown phosphatases can initiate cytosolic purine nucleotide catabolism. Here we show that Arabidopsis thaliana possesses a highly XMP-specific phosphatase (XMPP) which is conserved in vascular plants. We demonstrate that XMPP catalyzes the irreversible entry reaction of adenylate-derived nucleotides into purine nucleotide catabolism in vivo, whereas the guanylates enter catabolism via an unidentified GMP phosphatase and guanosine deaminase which are important to maintain purine nucleotide homeostasis. We also present a crystal structure and mutational analysis of XMPP providing a rationale for its exceptionally high substrate specificity, which is likely required for the efficient catalysis of the very small XMP pool in vivo

Analysis of Nucleosides and Nucleotides in Plants : An Update on Sample Preparation and LC–MS Techniques

Nucleotides fulfill many essential functions in plants. Compared to non-plant systems, these hydrophilic metabolites have not been adequately investigated in plants, especially the less abundant nucleotide species such as deoxyribonucleotides and modified or damaged nucleotides. Until recently, this was mainly due to a lack of adequate methods for in-depth analysis of nucleotides and nucleosides in plants. In this review, we focus on the current state-of-the-art of nucleotide analysis in plants with liquid chromatography coupled to mass spectrometry and describe recent major advances. Tissue disruption, quenching, liquid–liquid and solid-phase extraction, chromatographic strategies, and peculiarities of nucleotides and nucleosides in mass spectrometry are covered. We describe how the different steps of the analytical workflow influence each other, highlight the specific challenges of nucleotide analysis, and outline promising future developments. The metabolite matrix of plants is particularly complex. Therefore, it is likely that nucleotide analysis methods that work for plants can be applied to other organisms as well. Although this review focuses on plants, we also discuss advances in nucleotide analysis from non-plant systems to provide an overview of the analytical techniques available for this challenging class of metabolites

Analysis of Nucleosides and Nucleotides in Plants: An Update on Sample Preparation and LC–MS Techniques

Nucleotides fulfill many essential functions in plants. Compared to non-plant systems, these hydrophilic metabolites have not been adequately investigated in plants, especially the less abundant nucleotide species such as deoxyribonucleotides and modified or damaged nucleotides. Until recently, this was mainly due to a lack of adequate methods for in-depth analysis of nucleotides and nucleosides in plants. In this review, we focus on the current state-of-the-art of nucleotide analysis in plants with liquid chromatography coupled to mass spectrometry and describe recent major advances. Tissue disruption, quenching, liquid–liquid and solid-phase extraction, chromatographic strategies, and peculiarities of nucleotides and nucleosides in mass spectrometry are covered. We describe how the different steps of the analytical workflow influence each other, highlight the specific challenges of nucleotide analysis, and outline promising future developments. The metabolite matrix of plants is particularly complex. Therefore, it is likely that nucleotide analysis methods that work for plants can be applied to other organisms as well. Although this review focuses on plants, we also discuss advances in nucleotide analysis from non-plant systems to provide an overview of the analytical techniques available for this challenging class of metabolites