Signal-metabolome interactions in plants [Elektronische Ressource] / by Claudia Sabine Birkemeyer

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• Biologie

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• Publié par: universitat_potsdam
• Publié le: 01 janvier 2006
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Max-Planck-Institute of Molecular Plant Physiology
Department I, Root Metabolism
Signal-Metabolome Interactions in Plants
Dissertation
Submitted for graduation as
"Doctor rerum naturalium"
(Dr. rer. nat.)
in "Analytical Biochemistry"
A thesis submitted to the
Mathematisch-Naturwissenschaftlichen Fakultät
of the University of Potsdam
by
Claudia Sabine Birkemeyer
thPotsdam, 5 November 2005The work presented in this thesis was carried out between January 2001 and December
2004 at the Max-Planck-Institute of Molecular Plant Physiology in Potsdam, Germany.
Examiner 1: Prof. Dr. Lothar Willmitzer
Max-Planck-Institute of Molecular Plant Physiology, Potsdam/ University
of Potsdam, Germany
Examiner 2: Prof. Dr. Elmar Weiler
Institute of Plant Physiology, Ruhr-University of Bochum, Germany
Examiner 3: Prof. Dr. John Pickett
Rothamsted Research, Biochemistry Division, Harpenden, Hertfordshire,
United Kingdom
Examiner 4: Prof. Dr. Ivo Feussner
Department of Plant Biochemistry, Georg-August-University of Göttingen,
Germany
2Statutory Declaration
This Ph. D. thesis is the account of practical work completed between January 2001 and
December 2004 in the department of Prof. Willmitzer at the Max-Planck-Institute of
Molecular Plant Physiology, Potsdam, Germany. I affirm that the presented document is
the result of my own work, without involving illegitimate references, and has not been
submitted for any degree at any other university.
Eidesstattliche Erklärung
Diese Dissertation ist das Ergebnis der von Januar 2001 bis Dezember 2004 am Max-
Planck-Institut für Molekulare Pflanzenphysiologie in Potsdam, Deutschland,
durchgeführten praktischen Arbeiten. Ich versichere, die vorliegende Arbeit selbständig
und ohne unzulässige Hilfe angefertigt und keine anderen als die angegebenen Quellen und
Hilfsmittel benutzt zu haben. Ich versichere weiter, daß die Arbeit bei keiner anderen
Hochschule als der Universität Potsdam eingereicht wurde.
Potsdam, den 05. November 2005
(Claudia Birkemeyer)
3Abstract
Abstract
From its first use in the field of biochemistry, instrumental analysis offered a variety of
invaluable tools for the comprehensive description of biological systems. Multi-selective
methods that aim to cover as many endogenous compounds as possible in biological
samples use different analytical platforms and include methods like gene expression profile
and metabolite profile analysis.
The enormous amount of data generated in application of profiling methods needs to be
evaluated in a manner appropriate to the question under investigation. The new field of
system biology rises to the challenge to develop strategies for collecting, processing,
interpreting, and archiving this vast amount of data; to make those data available in form of
databases, tools, models, and networks to the scientific community.
On the background of this development a multi-selective method for the determination of
phytohormones was developed and optimised, complementing the profile analyses which
are already in use (Chapter I). The general feasibility of a simultaneous analysis of plant
metabolites and phytohormones in one sample set-up was tested by studies on the
analytical robustness of the metabolite profiling protocol. The recovery of plant
metabolites proved to be satisfactory robust against variations in the extraction protocol by
using common extraction procedures of phytohormones; thus, a joint extraction of
metabolites and hormones from plant tissue appears practicable (Chapter II).
Quantification of compounds within the context of profiling methods requires particular
scrutiny (Chapter II). In Chapter III, the potential of stable-isotope in vivo labelling as
normalisation strategy for profiling data acquired with mass spectrometry is discussed.
First promising results were obtained for a reproducible quantification by stable-isotope in
vivo labelling, which was applied in metabolomic studies.
In-parallel application of metabolite and phytohormone profile analysis to seedlings of the
model plant Arabidopsis thaliana exposed to sulfate limitation was used to investigate the
relationship between the endogenous concentration of signal elements and the ‘metabolic
phenotype’ of a plant. An automated evaluation strategy was developed to process data of
compounds with diverse physiological nature, such as signal elements, genes and
metabolites – all which act in vivo in a conditional, time-resolved manner (Chapter IV).
Final data analysis focussed on conditionality of signal-metabolome interactions.
4Table of Contents
Table of Contents
ABSTRACT ..................................................................................................................................................... 4
TABLE OF CONTENTS ................................................................................................................................ 5
TABLES ........................................................................................................................................................... 6
FIGURES ......................................................................................................................................................... 7
INTRODUCTION ........................................................................................................................................... 8
PHYTOHORMONE ANALYSIS.......................................................................................................................... 9
THE CONCEPT OF PROFILING METHODS ...................................................................................................... 11
SIGNAL - METABOLOME INTERACTIONS...................................................................................................... 13
CHAPTER I - PHYTOHORMONE PROFILING - METHOD DEVELOPMENT ............................... 15
COMPREHENSIVE CHEMICAL DERIVATISATION FOR GAS CHROMATOGRAPHY – MASS SPECTROMETRY-
BASED MULTI-TARGETED PROFILING OF THE MAJOR PHYTOHORMONES ................................................... 15
ABSTRACT................................................................................................................................................... 15
INTRODUCTION............................................................................................................................................ 15
EXPERIMENTAL ........................................................................................................................................... 17
RESULTS AND DISCUSSION........................................................................................................................... 23
CONCLUSIONS ............................................................................................................................................. 31
CHAPTER II - ROBUSTNESS OF METABOLITE EXTRACTION FROM PLANT TISSUE........... 33
DESIGN OF METABOLITE RECOVERY BY VARIATIONS OF THE METABOLITE PROFILING PROTOCOL ........... 33
ABSTRACT................................................................................................................................................... 33
INTRODUCTION............................................................................................................................................ 34
EXPERIMENTAL ........................................................................................................................................... 35
RESULTS AND DISCUSSION.......................................................................................................................... 41
CONCLUSION ............................................................................................................................................... 52
CHAPTER III - NORMALISATION OF PROFILING DATA ............................................................... 54
METABOLOME ANALYSIS: THE POTENTIAL OF IN VIVO LABELLING WITH STABLE ISOTOPES FOR
METABOLITE PROFILING ............................................................................................................................. 54
ABSTRACT................................................................................................................................................... 54
INTRODUCTION............................................................................................................................................ 54
ANALYTICAL APPROACHES OF METABOLOME ANALYSIS: GENERAL VARIANTS, PROPERTIES AND
APPLICATIONS............................................................................................................................................. 56
QUANTITATIVE METABOLITE PROFILING BY ISOTOPOMER MASS RATIOS................................................... 59
13NOVEL APPLICATIONS OF C-SATURATED MICROBIAL METABOLOMES .................................................... 61
GLOSSARY................................................................................................................................................... 63
CHAPTER IV - METABOLITE AND HORMONE PROFILING DATA: DEVELOPMENT OF AN
EVALUATION CONCEPT ......................................................................................................................... 65
AUTOMATED PATHWAY SEARCH USING TRANS