Handbook of Plant Metabolomics

Molecular Plant Biology Handbook Series

Edited by Wolfram Weckwerth and Günter Kahl

ISBN 978-3-527-32777-5

This is the newest title in the successful Molecular Plant Biology Handbook Series. Just like the other titles in the series this new book presents an excellent overview of different approaches and techniques in Metabolomics. Contributors are either from ivy-league research institutions or from companies developing new technologies in this dynamic and fast-growing field. With its approach to introduce current techniques in plant metabolomics to a wider audience and with many labs and companies considering to introduce metabolomics for their research, the title meets a growing market. The Kahl books are in addition a trusted brand for the plant science community and have always sold above ex-pectations.

Weckw

erth · Kahl (Eds.)

Wolfram Weckwerth is Professor and Founding Chair of the Department of Molecular Systems Biology (MOSYS) at the University of Vienna, Austria. He holds a diploma in chemistry and a PhD in biochemistry, both from the Technical University Berlin, Germany. His main research interests are:

• The development of genome-wide metabolomics and proteomics/phospho- proteomics technologies as elementary systems biology techniques, high throughput profiling (HTP) in systems biology, data integration; combining experimental approaches with multivariate statistics, pattern recognition, modeling of metabolism, and functional genome interpretation: “synergetics”• The genotype-environment-phenotype-equation: plant ecology and pheno- typic plasticity including stress, growth, developmental and nutritional physiology• Plant-microbe-interaction and medicinal plants• Green Systems Biology: application of systems biology approaches in eco- logy, evolution and biotechnology

Günter Kahl is Professor for Plant Molecular Biology at the Biocenter of Johann Wolfgang Goethe-University of Frankfurt/Main, Germany. After gaining his PhD in plant biochemistry, he spent two postdoctoral years at Michigan State University, East Lansing, USA, joining Professor Joe Varner, and at the California Institute of Technology, Pasadena, California, with Pro-fessor James Bonner. His main research interests are:

• Sequencing and analysis of fungal, plant and animal genomes• Transcriptome analysis in pro- and eukaryotic organisms• Technology development

Günter Kahl is the author of more than 250 scientific journal publications and several book publications including all titles of the successful Molecular Plant Biology Handbook Series, and currently holds the CSO position at GenXPro GmbH, a company specializing in novel technologies in genomics and transcriptomics, located at the Frankfurt Innovation Centre for Biotech-nology (FIZ), Frankfurt/Main, Germany.

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Edited by

Wolfram Weckwerth

and Günter Kahl

The Handbook of PlantMetabolomics

Titles of the Series “Molecular Plant Biology Handbook Series”

Kahl, G., Meksem, K. (eds.)

The Handbook of Plant Functional GenomicsConcepts and Protocols

2008

ISBN: 978-3-527-31885-8

Meksem, K., Kahl, G. (eds.)

The Handbook of Plant Mutation ScreeningMining of Natural and Induced Alleles

2010

ISBN: 978-3-527-32604-4

Meksem, K., Kahl, G. (eds.)

The Handbook of Plant Genome MappingGenetic and Physical Mapping

2005

ISBN: 978-3-527-31116-3

Related Titles

Harbers, M., Kahl, G. (eds.)

Tag-based Next Generation Sequencing2012

ISBN: 978-3-527-32819-2

Hirt, H. (ed.)

Plant Stress BiologyFrom Genomics to Systems Biology

2010

ISBN: 978-3-527-32290-9

Hayat, S., Mori, M., Pichtel, J., Ahmad, A. (eds.)

Nitric Oxide in Plant Physiology2010

ISBN: 978-3-527-32519-1

Kahl, G.

The Dictionary of Genomics, Transcriptomics and Proteomics2009

ISBN: 978-3-527-32073-8

Edited by Wolfram Weckwerth and Günter Kahl

The Handbook of Plant Metabolomics

The Editors

Prof. Dr. Wolfram WeckwerthUniversität WienMolekulare SystembiologieAlthanstr. 141090 WienAustria

Prof. Dr. Günter KahlMohrmühlgasse 363500 SeligenstadtGermany

Cover Legend

The cover picture presents some structures ofrepresentative phytochemicals andbiosynthetic pathways and enzymes ofArabidopsis thaliana, referred to in the chapter“Integrative analysis of secondary metabolismand transcript regulation in Arabidopsisthaliana” by Fumio Matsuda and Kazuki Saito(for further details see Chapter 9, Fig. 4). Thefigure was originally published in “Matsuda,F., et al. (2010) AtMeteEpress development: Aphytochemical atlas of Arabidopsisdevelopment. Plant Physiol, 152, 566–578),www.plantphysiol.org,# American Society ofPlant Biologists. The permission of the authorsto partly use their figure in a changed formatis greatly appreciated. Foto of Arabidopsis:# Vasiliy Koval, Fotolia.com

Limit of Liability/Disclaimer of Warranty:While the publisherand author have used their best efforts in preparing thisbook, they make no representations or warranties withrespect to the accuracy or completeness of the contents ofthis book and specifically disclaim any implied warranties ofmerchantability or fitness for a particular purpose. Nowarranty can be created or extended by sales representativesor written sales materials. The Advice and strategiescontained herein may not be suitable for your situation. Youshould consult with a professional where appropriate.Neither the publisher nor authors shall be liable for any lossof profit or any other commercial damages, including but notlimited to special, incidental, consequential, or otherdamages.

Library of Congress Card No.: applied for

British Library Cataloguing-in-Publication DataA catalogue record for this book is available from the BritishLibrary.

Bibliographic information published by the DeutscheNationalbibliothekThe Deutsche Nationalbibliothek lists this publication in theDeutsche Nationalbibliografie; detailed bibliographic dataare available on the Internet at <http://dnb.d-nb.de>.

#2013 Wiley-VCH Verlag GmbH & Co. KGaA, Boschstr. 12,69469 Weinheim, Germany

Wiley-Blackwell is an imprint of John Wiley & Sons, formedby the merger of Wiley’s global Scientific, Technical, andMedical business with Blackwell Publishing.

All rights reserved (including those of translation into otherlanguages). No part of this book may be reproduced in anyform – by photoprinting, microfilm, or any other means –nor transmitted or translated into a machine languagewithout written permission from the publishers. Registerednames, trademarks, etc. used in this book, even when notspecifically marked as such, are not to be consideredunprotected by law.

Print ISBN: 978-3-527-32777-5ePDF ISBN: 978-3-527-66989-9ePub ISBN: 978-3-527-66990-5mobi ISBN: 978-3-527-66991-2oBook ISBN: 978-3-527-66988-2

Cover Design Adam-Design, WeinheimTypesetting Thomson Digital, Noida, IndiaPrinting and Binding Markono Print Media Pte Ltd,Singapore

Printed in SingaporePrinted on acid-free paper

Dedicated to

Ulrich and Hannelore Weckwerthfor their endless sympathy, patience and guidance

Contents

Preface XVIIList of Contributors XIX

Part I Central Metabolism 1

1 Metabolic Profiling of Plants by GC–MS 3Camilla B. Hill and Ute Roessner

1.1 Introduction 31.2 Methods and Protocols 71.2.1 Sample Preparation 71.2.1.1 Sampling 71.2.1.2 Homogenization and Extraction 71.2.1.3 Procedure for Polar Extraction of Metabolites 81.2.2 Chemical Derivatization: Methoxymation and Silylation 91.2.2.1 Procedure for the Chemical Derivatization of Plant Extracts 91.2.3 GC–MS Analysis 101.2.3.1 Procedure to Acquire GC–MS Data 111.2.4 Data Preprocessing and Export 121.2.4.1 Procedure for Postacquisition Data Preprocessing 121.2.4.2 Data Analysis and Statistics 141.2.4.3 Procedure for Postacquisition Data Analysis 151.3 Applications of the Technology 151.4 Perspectives 17

References 18

2 Isotopologue Profiling – Toward a Better Understandingof Metabolic Pathways 25Wolfgang Eisenreich, Claudia Huber, Erika Kutzner, Nihat Knispel,and Nicholas Schramek

2.1 Introduction 252.2 Methods and Protocols to Determine Isotopologues 312.2.1 Mass Spectrometry 312.2.2 Protocols for Isotopologue Profiling by GC–MS 36

jVII

2.2.2.1 Protein-Bound Amino Acids 362.2.2.2 Metabolic Intermediates and Polar Products 372.2.2.3 Carbohydrates 372.2.3 NMR Spectroscopy 382.2.4 Protocols for Isotopologue Profiling by NMR 412.2.5 Deconvolution of Isotopologue Data 432.2.6 Expanding the Metabolic Space by Retrobiosynthetic Analysis 452.3 Applications 462.3.1 Experiments Using ½U-13C6�Glucose 462.3.2 Experiments Using 13CO2 472.4 Perspectives 53

References 54

3 Nuclear Magnetic Resonance Spectroscopy for PlantMetabolite Profiling 57Sonia van der Sar, Hye Kyong Kim, Axel Meissner, Robert Verpoorte,and Young Hae Choi

3.1 Introduction 573.2 Methods and Protocols 593.2.1 Sample Preparation 593.2.1.1 Harvesting Plant Material 603.2.1.2 Drying 603.2.1.3 Extraction 603.2.2 Data Acquisition 603.2.3 Standard 1H-NMR Spectroscopy 613.2.4 J-Resolved Spectroscopy 613.2.5 Data Analysis 613.3 Applications 623.3.1 1D 1H-NMR Spectroscopy 623.3.2 2D NMR Spectroscopy 633.3.2.1 J-Resolved Spectroscopy 653.3.2.2 COSYand TOCSY 673.3.2.3 HMBC and HMQC/HSQC 683.3.2.4 NOESYor ROESY (CAMELSPIN) 693.3.2.5 DOSY 693.3.3 Magic Angle Spinning 703.4 Perspectives 71

References 72

4 Comprehensive Two-Dimensional Gas Chromatographyfor Metabolomics 77Katja Dettmer, Martin F. Almstetter, Christian J. Wachsmuth,and Peter J. Oefner

4.1 Introduction 774.2 Methods and Protocols 81

VIIIj Contents

4.2.1 Instrumentation 814.2.2 Sample Preparation and Analysis 824.2.3 Data Processing 834.2.4 Metabolic Fingerprinting 834.2.5 Quantitative Analysis of Selected Metabolites 844.3 Applications of the Technology 854.3.1 Data Analysis 854.3.2 Literature 884.4 Perspectives 89

References 90

5 MALDI Mass Spectrometric Imaging of Plants 93Ale9s Svato9s and Hans-Peter Mock

5.1 Introduction 935.1.1 Sample Preparation 965.1.2 Data Acquisition 985.1.3 Data Processing 985.2 Methods and Protocols 995.2.1 Sample Preparation and Handling 995.2.1.1 Intact Tissues 995.2.1.2 Cryosectioning 995.2.2 Matrix Deposition 1005.2.2.1 Paintbrush (Figure 5.2) 1005.2.2.2 Sublimation (Figure 5.3) 1025.2.3 MALDI-MS Imaging Measurement 1035.2.3.1 Bruker Ultraflex Instruments 1035.2.3.2 Waters MALDI Micro MX 1045.3 Imaging Intact Tissues and Objects 1055.4 Future Perspectives 109

References 109

6 Medicago truncatula Root and Shoot Metabolomics: Protocolfor the Investigation of the Primary Carbon and Nitrogen MetabolismBased on GC–MS 111Vlora Mehmeti, Lena Fragner, and Stefanie Wienkoop

6.1 Introduction 1116.2 Methods and Protocols 1126.2.1 Equipment and Software 1126.2.2 Buffers and Chemicals 1126.2.3 Plant Material and Harvest 1136.2.4 Extraction 1146.2.5 Derivatization 1156.2.6 GC–MS Setup for the Analysis 1156.2.7 Metabolite Identification and Quantification: Data Matrix

Processing 116

Contents jIX

6.2.8 Data Mining 1196.3 Applications of the Technology 1196.4 Perspectives 121

References 123

Part II Secondary and Lipid Metabolism 125

7 Study of the Volatile Metabolome in Plant–Insect Interactions 127Georg J.F. Weingart, Nora C. Lawo, Astrid Forneck, Rudolf Krska,and Rainer Schuhmacher

7.1 Introduction 1277.1.1 Plant–Insect Interactions 1277.1.2 Significance of Volatile Plant Metabolites 1287.1.3 Study of the Plant Volatile Metabolome in Plant–Insect Interactions 1287.1.3.1 Setting Up of Biological Experiments 1297.1.3.2 Sampling, Quenching, and Sample Preparation 1307.1.3.3 Headspace Extraction and Measurement by GC–MS 1317.1.3.4 Data Handling 1347.1.3.5 Biological Interpretation 1357.2 Methods and Protocols 1357.2.1 Permanent Breed of Insects 1357.2.2 Cultivation of Grapevine Plants and Inoculation with Phylloxera 1367.2.2.1 Materials 1367.2.2.2 Procedures 1367.2.3 Sampling and Quenching of Plant Tissue (Roots and Leaves) 1387.2.3.1 Sampling and Quenching of Root Tips 1387.2.3.2 Sampling and Quenching of Grapevine Leaves 1397.2.4 Milling and Weighing of Plant Tissue (Roots and Leaves) 1407.2.4.1 Milling and Weighing of Root Samples 1407.2.4.2 Milling and Weighing of Leaf Samples 1417.2.5 Measurement – Automated HS-SPME Extraction

and GC–MS Analysis 1437.2.5.1 Materials 1437.2.5.2 SPME Method 1437.2.5.3 GC Method 1447.2.5.4 MS Settings 1447.2.6 Data Processing with AMDIS 1457.2.6.1 An In-House Reference Library Has to be Established in Advance 1457.2.6.2 Generation of RI Calibration File 1467.2.6.3 Batch Job Analysis for the Simultaneous Processing of Multiple

Sample Chromatograms 1467.2.7 Statistics/Chemometrics 1477.2.7.1 Univariate Statistics 1477.2.7.2 Multivariate Statistics 148

Xj Contents

7.3 Applications of the Technology 1487.4 Perspectives 149

References 150

8 Metabolomics in Herbal Medicine Research 155Lie-Fen Shyur, Chiu-Ping Liu, and Shih-Chang Chien

8.1 Introduction 1558.2 Methods and Protocols 1588.2.1 Materials 1588.2.1.1 Reagents 1588.2.1.2 Equipment 1598.2.2 Procedures 1608.2.2.1 Sample Handling for Medicinal Plants 1608.2.2.2 Sample Preparation for LC–MS Analysis 1608.2.2.3 LC–MS Analysis 1618.2.2.4 HPLC–Photodiode Array (PDA) MS Setup and Analysis 1618.2.2.5 GC–MS Analysis 1628.2.2.6 Plant Extract Preparation for GC–MS Analysis 1638.2.2.7 GC–MS Parameters and Analysis 1648.2.2.8 LC–MS and GC–MS Data Analysis 1658.2.2.9 LC–SPE–NMR Analysis 1668.2.2.10 Sample Preparation and LC–SPE–NMR Analysis 1678.2.2.11 HPLC–SPE–NMR Data Analysis 1688.3 Applications 1688.4 Perspectives 169

References 170

9 Integrative Analysis of Secondary Metabolism and TranscriptRegulation in Arabidopsis thaliana 175Fumio Matsuda and Kazuki Saito

9.1 Introduction 1759.2 Methods and Protocols 1779.2.1 Metabolome Analysis of Plant Secondary Metabolites 1779.2.1.1 Sample Preparation 1779.2.1.2 Data Acquisition 1789.2.1.3 Preparation of Metabolite Accumulation Data from the Raw

Chromatogram Data 1799.2.2 Preparation of Combined Data Matrix 1809.2.2.1 Preparation of Gene Expression Data 1809.2.2.2 Combination of Data Matrices 1809.2.3 Data Mining 1809.2.3.1 BL-SOM Analysis 1809.2.3.2 Correlation Analysis 1819.2.3.3 Principal Component Analysis and Application of Other

Data Mining Techniques 183

Contents jXI

9.3 Applications of the Technology 1839.4 Perspectives 187

References 190

10 Liquid Chromatographic–Mass Spectrometric Analysis ofFlavonoids 197Maciej Stobiecki and Piotr Kachlicki

10.1 Introduction 19710.1.1 Role of Flavonoids and Their Derivatives in Biological Systems 19710.1.2 Preparation of Biological Material for Metabolomic Analysis and/or

Metabolite Profiling 19910.1.3 Instrumental Considerations 20110.2 Methods and Protocols: Liquid Chromatography–Mass

Spectrometry of Flavonoids 20610.2.1 General Remarks 20610.2.2 Plant Cultivation Conditions 20810.2.3 Preparation of Biological Material with Biotechnological Methods

(Callus, Cell, or Hairy Root Cultures) 20810.2.4 Extraction of Plant Tissue or Biotechnologically Prepared Material 20810.2.4.1 Extraction Procedure 20910.2.5 Solid-Phase Extraction of Culture Medium or Apoplastic Fluids 20910.2.6 Preparation of Samples for LC–MS Analyses 21010.2.7 Chromatographic Protocols for Separation of Flavonoid

Glyconjugates 21010.2.8 Control of Ionization Parameters During Mass Spectrometric

Analysis and Identification of Compounds During LC–MSMetabolite Profiling 211

10.3 Applications of the Technology 21110.4 Perspectives 211

References 212

11 Introduction to Lipid (FAME) Analysis in Algae Using GasChromatography–Mass Spectrometry 215Takeshi Furuhashi and Wolfram Weckwerth

11.1 Introduction 21511.2 Methods and Experimental Protocol 21611.2.1 Extraction 21611.2.2 Bound and Free Fatty Acids 21711.2.3 Pigments 21711.2.4 Contaminants 21911.2.5 Derivatization 21911.2.6 GC–MS System 22011.2.7 Identification 22011.2.8 Protocols 22111.2.8.1 Protocol I 221

XIIj Contents

11.2.8.2 Protocol II 22111.2.9 GC–MS Instrument and Conditions 22311.3 Application and Perspective 223

References 224

12 Multi-Gene Transformation for Pathway Engineering of SecondaryMetabolites 227Hideyuki Suzuki, Eiji Takita, Kiyoshi Ohyama, Satoru Sawai, Hikaru Seki,Nozomu Sakurai, Toshiya Muranaka, Masao Ishimoto, Hiroshi Sudo,Kazuki Saito, and Daisuke Shibata

12.1 Introduction 22712.2 Methods and Protocols 23312.2.1 Chemicals 23312.2.2 Plasmid Construction of Multi-Gene Transformation 23312.2.3 Preparation of Dual Terminator (DT) Fragment by PCR-Based

Overlap Extension Method 23312.2.4 Plasmid Construction of pUHR KS CSPS Thsp 23612.2.5 Construction of pHSG299 CSPS 35S-CYP88-DT (Figure 12.2a) 23612.2.6 Construction of pHSG299 CSPS 35S-CYP72-DT2 (Figure 12.2a) 23712.2.7 Construction of pHSG299-CYP93(RNAi)-DT (Figure 12.2a) 23812.2.8 Construction of pUHR KS CSPS Thsp-CYP88-CYP72-CYP93

(RNAi) 23912.2.9 Transformation of Soybean by Particle Bombardment 23912.2.9.1 Preparation of Embryogenic Suspension Tissue Culture 23912.2.9.2 Preparation of Plasmid DNA for Particle Bombardment 24012.2.9.3 Conditions of Particle Bombardment 24012.2.9.4 Selection and Generation of Transgenic Soybean Plants 24012.2.10 GC-MS Analysis for Triterpene Glycone 24112.2.10.1 Extraction of Metabolite 24112.2.10.2 Acid Treatment of Extracted Metabolites 24112.2.10.3 Derivatization of Metabolites 24212.2.11 GC-MS Conditions 24212.3 Application of Technology 24212.4 Perspectives 243

References 243

Part III Metabolomics and Genomics 245

13 Metabolomics-Assisted Plant Breeding 247Alexander Herrmann and Nicolas Schauer

13.1 Introduction 24713.2 Method 24913.3 Applications of the Technology 25113.4 Perspective 253

References 254

Contents jXIII

14 Conducting Genome-Wide Association Mapping of Metabolites 255Susanna Atwell and Daniel J. Kliebenstein

14.1 Introduction 25514.2 Methods and Protocols 25614.2.1 Biological Question to Be Addressed 25614.2.2 Chemistry to Study 25614.2.2.1 Chemical Class 25614.2.2.2 Extraction and Detection Platform 25714.2.3 Species Choice 25814.2.3.1 Genotypic Choices 25814.2.3.2 GWA Populations Available 25914.2.3.3 Domestication Status 26014.2.3.4 Ability to Conduct Appropriate Follow-Up Experiments 26014.2.4 Should I Utilize an Additional Perturbation? 26014.2.5 Conducting the Phenotype Measurements 26114.2.6 Computational Platform to Use for Analysis 26114.2.6.1 Single Marker Analysis 26214.2.6.2 Population Structure Modification 26214.2.6.3 Resulting GWA Plots 26214.2.6.4 Gene-Based Approaches 26314.2.6.5 What Should I Use and How Do I Use It? 26314.2.7 Candidate Gene Selection 26514.2.8 Candidate Gene Validation 26614.2.8.1 Validate That the Gene Influences the Phenotype? 26714.2.8.2 Validate That Natural Variation in the Gene Influences the

Phenotype 26714.3 Applications 26714.4 Perspectives 268

References 268

Part IV Metabolomics and Bioinformatics 273

15 Metabolite Clustering and Visualization of Mass Spectrometry DataUsing One-Dimensional Self-Organizing Maps 275Alexander Kaever, Manuel Landesfeind, Kirstin Feussner, Ivo Feussner,and Peter Meinicke

15.1 Introduction 27515.2 Methods and Protocols 27615.2.1 Data Import 27715.2.2 Clustering 27715.2.3 Cluster Analysis 28015.3 Applications of the Technology 28115.4 Perspectives 286

References 286

XIVj Contents

16 Metabolite Identification and Computational Mass Spectrometry 289Steffen Neumann, Florian Rasche, Sebastian Wolf, and Sebastian B€ocker

16.1 Introduction 28916.2 Annotation and Identification of Metabolites 29016.2.1 Exact Mass Search in Compound Libraries 29116.2.2 Deriving the Elemental Composition from MS1 29216.2.3 Elemental Composition from MS2 and MSn 29316.2.4 In Silico Library Search with MetFrag 29416.2.5 Reference Spectral Library Lookup 29916.3 Perspectives 302

References 303

17 Using COVAIN to Analyze Metabolomics Data 305Xiaoliang Sun and Wolfram Weckwerth

17.1 Introduction 30517.2 Methods 30817.2.1 Data Preprocessing 30817.2.1.1 Imputation of Missing Values 30817.2.1.2 Transformations to Satisfy Prerequisites of Statistical Methods 31017.2.1.3 Adjusting Outliers 31017.2.1.4 Scaling 31017.2.1.5 Filtering by Statistical Features 31017.2.2 Uni- and Bivariate Statistical Methods for Individual

Metabolite-Level Analysis 31117.2.2.1 ANOVA Compares Single Metabolite Levels 31117.2.2.2 Correlation Coefficients Interpret the Relationships Between

Pairwise Two Metabolites 31117.2.2.3 Granger Causality Analysis Identifies the Causation Between

Pairwise Two Metabolites in Time-Series Data 31117.2.3 Multivariate Statistical Methods for Group-Level Analysis 31217.2.3.1 PCA Distinguishes Phenotypes and Finds Most Influencing

Metabolites 31217.2.3.2 Independent Component Analysis Distinguishes Phenotypes

and Finds the Latent Sources of Metabolites in Time-Series Data 31217.2.3.3 Clustering Classifies Data Into Groups 31217.2.4 Network-Level Analysis 31317.2.4.1 Network Mapping 31317.2.4.2 Network Inference 31317.2.5 Influences of Data Preprocessing on Statistical Analysis Results 31317.2.5.1 On the Mean Values: ANOVA, Correlation Coefficient, Granger

Analysis, and Clustering 31317.2.5.2 On the Variance and Covariance: ANOVA, PCA, and ICA 31417.3 Application 31417.4 Perspective 320

References 320

Contents jXV

18 Mass Spectral Search and Analysis Using the Golm MetabolomeDatabase 321Jan Hummel, Nadine Strehmel, Christian B€olling, Stefanie Schmidt,Dirk Walther, and Joachim Kopka

18.1 Introduction 32118.2 Methods and Protocols: the GMD and Supported Data Analysis

Workflows 32218.2.1 The GMD Data Entities 32218.2.2 The Text Search Queries 32518.2.3 The Mass Spectrum Query Submission and Analysis Options 32518.2.3.1 Mass Spectral Matching 32618.2.3.2 Decision Tree (DT)-Supported Substructure Prediction 32918.2.4 Interpreting the Mass Spectral Analysis Results 32918.2.4.1 The Mass Spectral Matching Results 32918.2.4.2 The Substructure Prediction Results 33218.2.4.3 Interpreting Decision Trees 33318.2.5 The Web Services at GMD 33618.2.5.1 General Considerations 33618.2.5.2 The GMDWeb Service Modules 33718.2.6 The GMD Download Options 33818.3 Applications and Perspectives 341

References 342

Glossary 345

Index 415

XVIj Contents

Preface

Whereas the most modern topics of plant research, grouped into the term “omics,”such as genomics, transcriptomics, and proteomics, are comparatively new, thestrategies to isolate, purify, identify, and quantify a multitude of low molecularweight cellular compounds (called metabolites) look back onto a long history. Whileinitially only metabolites present in comparatively high concentrations could beisolated and quantified (sometimes only semiquantified), the advent of enzymaticdetection techniques in the 1970s brought a breakthrough in precise metaboliteanalysis. Unfortunately, these techniques required the coupling of a metabolite’sdetection to the reduction/oxidation of NADþ or NADPþ, and therefore excludedthe majority of metabolites, especially secondary metabolites. More recent develop-ments in mass spectrometry (MS), matrix-assisted laser desorption/ionization(MALDI)–MS for metabolite imaging, gas chromatography coupled to mass spec-trometry (GC–MS), liquid chromatography coupled to mass spectrometry (LC–MS),and NMR technology for medium- to high-throughput identification and quantifi-cation of low molecular weight compounds pushed metabolite analysis to today’sadvanced level, where various physico-chemical separation techniques are com-bined to analyze metabolite profiles in considerable detail and accuracy. The presentstate of technology for metabolite analysis has been denoted “metabolomics.”Metabolomics reflects the physiological state of an organism or its organs, tissues,

or cells, and therefore allows a hitherto not possible comprehensive understandingof the biology of an organism and its response to intrinsic or environmental changesor influences. The various techniques of metabolomics allow the comprehensiveprofiling of cellular metabolites at the systems level, thereby providing a directreadout of biochemical activity that can be correlated with phenotype and used toidentify therapeutic targets. This omics discipline then bridges the gap betweengenotype and phenotype. The present Handbook of Plant Metabolomics not onlywitnesses the present state-of-the-art metabolomics and its widespread applications,but also portrays up-to-date technical advances in metabolic fingerprinting and thein silico analysis of the resulting, mostly very complex, metabolite patterns.The Handbook of Plant Metabolomics (Metabolite Profiling and Networking) is the

fourth volume in the successful Wiley-VCH series of Handbooks of Plant GenomeAnalysis, and follows the warmly welcomed The Handbook of Plant Genome Mapping(Genetic and Physical Mapping), The Handbook of Plant Functional Genomics

jXVII

(Concepts and Protocols), and The Handbook of Plant Mutation Screening (Mining ofNatural and Induced Alleles). It provides informative introductions to each chapter,detailed descriptions of techniques for metabolite profiling, and robust and ready-to-go laboratory protocols, in addition to some applications, all written by internation-ally renowned experts in their research fields. Although this volume focuses on plantmetabolomics, the techniques presented are broadly applicable to other biologicalsystems exemplifying the pioneering and original character of metabolomics inplant biology. This rapid development of metabolomics to a mature technology iscatalyzing the application of metabolomics in other fields of research also, such asbiomedicine.The Editors very much appreciate the excellent chapters contributed by all the

authors, and expect that The Handbook of Plant Metabolomics will reproduce theworldwide success of its three progenitors.

Vienna (Austria) Wolfram WeckwerthFrankfurt am Main (Germany) G€unter KahlAugust 2012

XVIIIj Preface

List of Contributors

Martin F. AlmstetterUniversity of RegensburgInstitute of Functional GenomicsJosef-Engert-Strasse 993053 RegensburgGermany

Susanna AtwellUniversity of California, DavisDepartment of Plant SciencesOne Shields AvenueDavis, CA 95616USA

Sebastian B€ockerFriedrich-Schiller-University JenaInstitute for InformaticsErnst-Abbe-Platz 207743 JenaGermany

Christian B€ollingCharit�e Universit€atsmedizin BerlinComputational SystemsBiochemistry GroupSeestrasse 7313347 BerlinGermany

Shih-Chang ChienNational Chung Hsing UniversityExperimental Forest ManagementOfficeNo. 250, Kuo Kuang RoadTaichung 402Taiwan

Young Hae ChoiLeiden UniversityInstitute of BiologyNatural Products LaboratorySylviusweg 72, 2333 BELeidenThe Netherlands

Katja DettmerUniversity of RegensburgInstitute of Functional GenomicsJosef-Engert-Strasse 993053 RegensburgGermany

Wolfgang EisenreichTechnische Universit€at M€unchenLehrstuhl f€ur BiochemieLichtenbergstrasse 485435 GarchingGermany

jXIX

Ivo FeussnerGeorg-August-Universität GöttingenAlbrecht-von-Haller-Institute forPlant SciencesDepartment of Plant BiochemistryJustus-von-Liebig-Weg 1137077 GöttingenGermany

Kirstin FeussnerGeorg-August-Universität GöttingenInstitute of Microbiology andGeneticsDepartment of MolecularMicrobiology and GeneticsGrisebachstrasse 837077 GöttingenGermany

and

Georg-August-Universität GöttingenAlbrecht-von-Haller-Institute forPlant SciencesDepartment of Plant BiochemistryJustus-von-Liebig-Weg 1137077 GöttingenGermany

Astrid ForneckUniversity of Natural Resources andLife Sciences, ViennaDepartment of Crop SciencesDivision of Viticulture and PomologyKonrad-Lorenz-Strasse 243430 TullnAustria

Lena FragnerUniversity of ViennaDepartment of Molecular SystemsBiologyAlthanstrasse 141090 ViennaAustria

Takeshi FuruhashiUniversity of ViennaDepartment of Molecular SystemsBiology (MOSYS)Althanstrasse 141090 ViennaAustria

Alexander HerrmannMetabolomic Discoveries GmbHAm M€uhlenberg 1114476 Potsdam-GolmGermany

Camilla B. HillThe University of MelbourneSchool of Botany, Building 122Australian Centre for PlantFunctional Genomics (ACPFG)Professors WalkParkville, VIC 3052Australia

Claudia HuberTechnische Universit€at M€unchenLehrstuhl f€ur BiochemieLichtenbergstrasse 485435 GarchingGermany

Jan HummelMax Planck Institute of MolecularPlant Physiology (MPIMP)Bioinformatics GroupAm Muehlenberg 114476 Potsdam-GolmGermany

Masao IshimotoNational Institute of AgrobiologicalSciences2-1-2 KannondaiTsukubaIbaraki 305-8602Japan

XXj List of Contributors

Piotr KachlickiInstitute of Plant Genetics PASStrzeszy�nska 3460-479 Pozna�nPoland

Alexander KaeverGeorg-August-Universität GöttingenInstitute of Microbiology andGeneticsDepartment of BioinformaticsGoldschmidtstrasse 137077 GöttingenGermany

Hye Kyong KimLeiden UniversityInstitute of BiologyNatural Products LaboratorySylviusweg 72, 2333 BELeidenThe Netherlands

Daniel J. KliebensteinUniversity of California, DavisDepartment of Plant SciencesOne Shields AvenueDavis, CA 95616USA

Nihat KnispelTechnische Universit€at M€unchenLehrstuhl f€ur BiochemieLichtenbergstrasse 485435 GarchingGermany

Joachim KopkaMax Planck Institute of MolecularPlant PhysiologyApplied Metabolome AnalysisDepartment 1Prof. Lothar WillmitzerAm Muehlenberg 1D-14476 Potsdam-GolmGermany

Rudolf KrskaUniversity of Natural Resources andLife Sciences, ViennaCenter for Analytical ChemistryDepartment IFA-TullnKonrad-Lorenz-Strasse 203430 TullnAustria

Erika KutznerTechnische Universit€at M€unchenLehrstuhl f€ur BiochemieLichtenbergstrasse 485435 GarchingGermany

Manuel LandesfeindGeorg-August-Universität GöttingenInstitute ofMicrobiology andGeneticsDepartment of BioinformaticsGoldschmidtstrasse 137077 GöttingenGermany

Nora C. LawoUniversity of Natural Resources andLife Sciences, ViennaDivision of Viticulture and PomologyDepartment of Crop SciencesKonrad-Lorenz-Strasse 243430 TullnAustria

and

Syngenta Crop ProtectionResearch SteinSchaffhauserstrasse 1014332 SteinSwitzerland

List of Contributors jXXI

Chiu-Ping LiuNational Taiwan UniversityInstitute of BiotechnologyNo. 1, Sec. 4, Roosevelt RoadTaipei 10617Taiwan

Fumio MatsudaRIKEN Plant Science CenterMetabolomicFunctionResearchGroupSuehiro-cho 1-7-22Tsurumi-kuYokohama 230-0045Japan

and

Osaka UniversityGraduate School of InformationScience and TechnologyDepartment of BioinformaticEngineering1-5, Yamada-oka SuitaOsaka 565-0871Japan

Vlora MehmetiUniversity of ViennaDepartment of Molecular SystemsBiologyAlthanstrasse 141090 ViennaAustria

Peter MeinickeGeorg-August-Universität GöttingenInstitute of Microbiology andGeneticsDepartment of BioinformaticsGoldschmidtstrasse 137077 GöttingenGermany

Axel MeissnerLeiden University Medical CenterDepartment of ParasitologyP.O. Box 9600Eindhovenweg 20, 2333 ZCLeidenThe Netherlands

Hans-Peter MockLeibniz Institute of Plant Geneticsand Crop Plant Research (IPK)Corrensstrasse 306466 GaterslebenGermany

Toshiya MuranakaYokohama City UniversityKihara Institute for BiologicalResearch641-12 Maioka-choTotsuka-kuYokohamaKanagawa 244-0813Japan

and

Osaka UniversityDepartment of Biotechnology2-1 YamadaokaSuita-shiOsaka 565-0871Japan

Steffen NeumannLeibniz Institute of PlantBiochemistry, IPB HalleDepartment of Stress andDevelopmental BiologyWeinberg 306120 Halle (Saale)Germany

XXIIj List of Contributors

Peter J. OefnerUniversity of RegensburgInstitute of Functional GenomicsJosef-Engert-Strasse 993053 RegensburgGermany

Kiyoshi OhyamaTokyo Institute of TechnologyGraduate School of Engineering2-12-1 OhokayamaMeguro-kuTokyo 152-8551Japan

and

RIKEN Plant Science Center1-7-22 Suehiro-choTsurumi-kuYokohamaKanagawa 230-0045Japan

Florian RascheFriedrich-Schiller-University JenaInstitute for InformaticsErnst-Abbe-Platz 207743 JenaGermany

Ute RoessnerThe University of MelbourneSchool of Botany, Building 122Australian Centre for PlantFunctional Genomics (ACPFG) andMetabolomics AustraliaProfessors WalkParkville, VIC 3052Australia

Kazuki SaitoRIKEN Plant Science CenterMetabolomic Function ResearchGroupSuehiro-cho 1-7-22Tsurumi-kuYokohama 230-0045Japan

and

Graduate School of PharmaceuticalSciencesDepartment ofMolecular Biology andBiotechnologyChiba UniversityInohana 1-8-1Chuo-kuChiba 260-8675Japan

Nozomu SakuraiKazusa DNA Research Institute2-6-7 Kazusa-KamatariKisarazuChiba 292-0818Japan

Satoru SawaiChiba UniversityGraduate School of PharmaceuticalSciences1-33 Yayoi-choInage-kuChiba 263-8522Japan

and

Tokiwa Phytochemical Co., Ltd.58 KinokoSakuraChiba 285-0801Japan

List of Contributors jXXIII

and

RIKEN Plant Science Center1-7-22 Suehiro-choTsurumi-kuYokohamaKanagawa 230-0045Japan

Nicolas SchauerMetabolomic Discoveries GmbHAm M€uhlenberg 1114476 Potsdam-GolmGermany

Stefanie SchmidtMax Planck Institute of MolecularPlant PhysiologyApplied Metabolome AnalysisDepartment 1Prof. Lothar WillmitzerAm Muehlenberg 1D-14476Potsdam-GolmGermany

Nicholas SchramekTechnische Universit€at M€unchenLehrstuhl f€ur BiochemieLichtenbergstrasse 485435 GarchingGermany

and

Bavarian Health and Food SafetyAuthorityVeterin€arstrasse 285764 OberschleissheimGermany

Rainer SchuhmacherUniversity of Natural Resources andLife Sciences, ViennaCenter for Analytical ChemistryDepartment IFA-TullnKonrad-Lorenz-Strasse 203430 TullnAustria

Hikaru SekiYokohama City UniversityKihara Institute for BiologicalResearch641-12 Maioka-choTotsuka-kuYokohamaKanagawa 244-0813Japan

and

Osaka UniversityDepartment of Biotechnology2-1 YamadaokaSuita-shiOsaka 565-0871Japan

Daisuke ShibataKazusa DNA Research Institute2-6-7 Kazusa-KamatariKisarazuChiba 292-0818Japan

Lie-Fen ShyurAgricultural Biotechnology ResearchCenterAcademia SinicaNo. 128, Sec. 2, Academia RoadNankangTaipei 115Taiwan

XXIVj List of Contributors

Maciej StobieckiInstitute of Bioorganic ChemistryPASNoskowskiego 12/14Laboratory of Natural ProductsBiochemistry61-704 Pozna�nPoland

Nadine StrehmelLeibniz Institute of PlantBiochemistry, IPB HalleDepartment of Stress andDevelopmental BiologyWeinberg 306120 Halle (Saale)Germany

Hiroshi SudoChiba UniversityGraduate School of PharmaceuticalSciences1-33 Yayoi-choInage-kuChiba 263-8522Japan

and

Tokiwa Phytochemical Co., Ltd.58 KinokoSakuraChiba 285-0801Japan

and

Hoshi UniversitySchool of Pharmacy andPharmaceutical Sciences2-4-41 EbaraShinagawa-kuTokyo 142-8501Japan

Xiaoliang SunUniversity of ViennaDepartment of Molecular SystemsBiologyAlthanstrasse 141090 ViennaAustria

Hideyuki SuzukiKazusa DNA Research Institute2-6-7 Kazusa-KamatariKisarazuChiba 292-0818Japan

Ale9s Svato9sMax Planck Institute for ChemicalEcologyMass Spectrometry Research GroupHans-Knoell-Strasse 807745 JenaGermany

Eiji TakitaKazusa DNA Research Institute2-6-7 Kazusa-KamatariKisarazuChiba 292-0818Japan

and

Idemitsu Kosan Co., Ltd.1280 KamiizumiSodegaura-shiChiba 299-0293Japan

Sonia van der SarLeiden UniversityInstitute of BiologyNatural Products LaboratorySylviusweg 72, 2333 BELeidenThe Netherlands

List of Contributors jXXV

Robert VerpoorteLeiden UniversityInstitute of BiologyNatural Products LaboratorySylviusweg 72, 2333 BELeidenThe Netherlands

Christian J. WachsmuthUniversity of RegensburgInstitute of Functional GenomicsJosef-Engert-Strasse 993053 RegensburgGermany

Dirk WaltherMax Planck Institute of MolecularPlant Physiology (MPIMP)Bioinformatics GroupAm Muehlenberg 114476 Potsdam-GolmGermany

Wolfram WeckwerthUniversity of ViennaDepartment of Molecular SystemsBiologyAlthanstrasse 141090 ViennaAustria

Georg J.F. WeingartUniversity of Natural Resources andLife Sciences, ViennaCenter for Analytical ChemistryDepartment IFA-TullnKonrad-Lorenz-Strasse 203430 TullnAustria

and

Fondazione Edmund MachResearch and Innovation CentreFood Quality and NutritionDepartmentVia E. Mach 138010 San Michele all’Adige (TN)Italy

Stefanie WienkoopUniversity of ViennaDepartment of Molecular SystemsBiologyAlthanstrasse 141090 ViennaAustria

Sebastian WolfLeibniz Institute of PlantBiochemistry, IPB HalleDepartment of Stress andDevelopmental BiologyWeinberg 306120 Halle (Saale)Germany

XXVIj List of Contributors

Part ICentral Metabolism

The Handbook of Plant Metabolomics, First Edition. Edited by Wolfram Weckwerth and Günter Kahl# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.

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